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LECTURE 1
Of Special Course “Modern Problems of Molecular Biology”
for Pharmaceutical Department students of the 2nd Year of Study
Theme:
“MOLECULAR BIOLOGY: SUBJECT AND TASKS. DNA STRUCTURE,
FUNCTIONS AND PROPERTIES. MOLECULAR MECHANISMS OF DNA
REPLICATION, RECOMBINATION AND REPAIR. MOLECULAR STRUCTURE
OF A GENE. STRUCTURE OF GENOMES OF VIRUSES, PRO- AND
EUKARYOTES”
Lecture plan:
1. Subject, aim and tasks of Molecular Biology Course
2. Steps of Molecular Biology development
3. DNA chemical structure, macromolecular arrangement, functions and properties
3.1 Form, structure and macromolecular properties of DNA
3.2 Primary DNA structure:
3.3 DNA chemical characteristic
3.4 Secondary structure of DNA
3.5 Tertiary structure of DNA: double helix
3.6 DNA conformations
4. DNA replication
5. DNA self-correction
6. DNA damage
7. DNA reparation
8. DNA and RNA comparison. RNA types and properties
9. Gene definition and classification
9.1 Gene types
9.2 Gene properties
10.Sequences of human genome
10.1 Regulatory sequences
10.2 Repetitive sequences
10.3 Gene clusters
10.4 Pseudogenes
10.5 Tandemly repeated (satellite) DNA
11. Transposed sequences
11.1 Transposons causing diseases
12. Non-nuclear heredity
12.1 Mitochondrial genome
1. Subject, aim and tasks of Molecular Biology Course
All living beings have molecular basis of structure and functions.
The bodies of living organisms consist of approximately 30 main elements. They are (a
99% of mass) carbon, oxygen, hydrogen, nitrogen mostly and phosphorus and sulfur as well.
Sodium, potassium, iron, calcium, magnesium, chlorine and iodine are compounds of a
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great importance also.
The atoms of the various elements are «combined» in different proportions and constitute
the enormous amount of the varied molecules are creating the living systems (cells).
Molecules are compose living beings are subdivided into organic and inorganic
The inorganic molecules of living beings are represented by water (70% from body
mass), and different solved salts.
Compound organic molecules have significant weight. They contain carbon as a main
component and also include hydrogen, oxygen, nitrogen, phosphorus and sulfur. The organic
polymers made from amino acids compose proteins’ molecules. Polymers are composed from
nucleotides have an important role in energy accumulation and transduction (ATP, GTP and
others). However, main quantity of nucleotides are represented in a cell are subunits of
informative molecules DNA and RNA.
Thus, a living being can be defined as the highly-organized arrangement of molecules with the
permanent usage of energy and matter of surrounding environment «under the direction of» the
informative programs of nucleic acids. They provide the prolongated support of high efficiency
arrangement of molecules and processes in living beings.
1.1.Molecular Biology subject and tasks:
Molecular biology is science about the mechanisms of genetic information storage,
transmission and expression, about intermolecular interaction which underlaid biological
processes, about a structure and functions of irregular biopolimers - nucleic acids and proteins.
All molecules of the living systems are the subjects of molecular biology.
Tasks of Molecular Biology are following:
1) Study of molecular structures of the living systems
2) Study of relations between the molecular structures of living beings
3) Study of genes and their functions
4) Application of the knowledge for molecular biotechnology
The main aim of the studying the Molecular Biology’ Subject is:
To be able to explain vital functions of human organism at molecular-genetic level of structural
organization.
2. Steps of Molecular Biology development
1902 - Walter Sutton created term "gene" to describe "factors“ located on chromosomes: he
observed chromosomal movement during meiosis and developed the chromosomal theory
of heredity
1905-1908 - William Bateson and Reginal Crudell Punnett demonstrated actions of some genes
modify action of other genes: the first time gene regulation was demonstrated
1933 - A new technique, electrophoresis, was introduced by Arne Tiselius for separating
proteins in solution
1937 - Frederick Charles Bawden discovered tobacco mosaic virus RNA.
1944 - Barbara McClintock reported transposable elements: "jumping genes"
1946 - Edward Tatum and Joshua Lederberg discovered that bacteria can exchange genetic
material directly through conjugation. Max Delbruck and Alfred Day Hershey discovered a
combination of genetic material from viruses: genetic recombination
1950 - Erwin Chargaff found that amounts of adenine and thymine and cytosine and guanine in
DNA are always about the same. This is now called "Chargaff's Rules"
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1953 - James Watson and Francis Crick proposed the double-stranded, helical, complementary,
anti-parallel model for DNA
1955 - Frederick Sanger announced the first complete sequence of a protein, bovine insulin
Arthur Kornberg discovered and isolated DNA polymerase from E. coli bacteria
1956 - Francis Crick and George Gamov worked out the "Central Dogma" to explain protein
synthesis from DNA: the DNA sequence codes for amino acid sequences and genetic
information flows in one direction - from DNA to mRNA to protein
1959 - Francois Jacob and Jacques Monod discovered an important mechanism behind genetic
regulation: mappable control functions located on chromosomes in DNA sequence - named
"repressor" and "operon"
1961 - Marshall Nirenberg, Heinrich Mathaei and Severo Ochoa cracked the "Genetic Code": a
sequence of three nucleotide bases (codon) determine each of amino acids
1967 Mary Weiss and Howard Green found a technique for combining human cells and mouse
cells grown in one culture: somatic cell hybridisation The first evolutionary trees from
protein sequences were set op by WM Fitch and E Margoliash
1970 - Howard Temin and David Baltimore independently isolated reverse transcriptase, an
enzyme that can make DNA from RNA
1972 - Paul Berg used a restriction enzyme to cut DNA and ligase to past two DNA strands
together to form hybrid circular molecule. This was the first recombinant DNA molecule
First successful DNA cloning experiments
1973 - Stanley Cohen and Herbert Boyer first successfully transfered DNA from one life form
into another: a spliced viral DNA and bacterial DNA to create a plasmid with dual
antibiotic resistance
1974 - Allan Maxam and Walter Gilbert (Harvard) and Frederick Sanger (U.K. Medical
Research Council) independently developed different methods for sequencing DNA
1977 - Bacteriophage FX-174 (5368 bp) was the first complete genome (DNA) to be sequenced
Richard Roberts’ and Phil Sharp’s labs showed that eukaryotic genes contain many
interruptions, called introns.
1978 - Genentech successfully produced human insulin using recombinant DNA technology in
E. coli David Botstein discovered the use of restriction enzymes produces different
fragments from one person to another, RFLP: restriction fragment length polymorphisms
1980 - Kary Mullis invented the polymerase chain reaction (PCR), a method for multiplying
DNA sequences in vitro
1981 - Gordon and Ruddle (Ohio University) made the first transgenic mice by inserting genes
from other animals with DNA microinjection. Human mitochondral DNA sequenced
(16569 bp)
1983 - First genetic modifed plant is created; a tobacco plant resistant to an antibiotic
1984 - Alec Jeffreys developed the technique of using sequences of DNA for identification,
called "genetic fingerprinting" Chiron Corp determined the entire sequence of the HIV-1
genome
1990 - Human Genome Project launched: estimated cost of $13 billion (plan 15 years) BLAST:
fast sequence similarity searching tool introduced by S. Karlin and S.F. Altshul
1994 - The FlavrSavr Tomato becomes the first genetic modified food to be approved for sale.
A gene expression the enzyme polygalacturonase, which is responsible for the tomato's
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softness, was introduced by Calgene
1999 - Drosophila melanogaster (fruitfly) genome completely sequenced (175 Mb)
2000 - Completion of the Arabidopsis thaliana sequence (157 Mb) Human genome draft
version finished (3200 Mb)
2010 Completion of the 2010 Project: the understanding the function of all genes within their
cellular, organismal and evolutionary context of Arabidopsis thaliana
Future goals of molecular biology and bioinformatics research
2050 - Completion of the first computational model of a complete cell, or maybe even already
of a complete organism
3. DNA chemical structure, macromolecular arrangement, functions and
properties
A nucleic acid is a complex, high-molecular-weight biochemical macromolecule
composed of smaller units nucleotides which created the chains that convey genetic
information.
Deoxyribonucleic acid (DNA) is a nucleic acid that contains the genetic instructions used
in the development and functioning of all known living organisms and some viruses. The main
role of DNA molecules is the long-term storage of information.
3.1 Form, structure and macromolecular properties of DNA
- DNA usually occurs as linear chromosomes in eukaryotes, and circular chromosomes in
prokaryotes.
- at most organisms DNA is a double helix; some viruses are exception, they have singlestranded DNA
- DNA has primary, secondary and tertiary levels of its structure;
- Prokaryotes have just 1 chromosome is represented with a single supercoiled DNA
macromolecule;
- Eukaryotic cells have DNA as a part of desoxyribonucleic complex (chromatin)
3.2 Primary DNA structure:
- it is the unramified polynucleotide chain with the certain sequence of nucleotides;
- is strictly specie-dependent and individual;
- represents the codified form of genetic information (genetic code);
- in the case of circular DNA the ends of molecule are reserved.
3.3 DNA chemical characteristic
The nucleotide repeats contain both the segment of the backbone of the molecule, which
holds the chain together, and a base, which interacts with the other DNA strand in the helix.
A base linked to a sugar is called a nucleoside and a base linked to a sugar and one or
more phosphate groups is called a nucleotide.
If multiple nucleotides are linked together, as in DNA, this polymer is called a
polynucleotide.
The backbone of the DNA strand is made from alternating phosphate and sugar residues.
The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar.
The sugars are joined together by phosphate groups that form phosphodiester bonds
between the third and fifth carbon atoms of adjacent sugar rings.
3.4 Secondary structure of DNA
- connected with a hydrogen bonds antiparallel, polynucleotide chains
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- each type of base on one strand forms a bond with just one type of base on the other strand in
accordance with Chargaffs’ rules. This is called complementary base pairing.
- different functions of complementary chains: coding chain and template chain
- efficiency of replication and transcription processes
3.5 Tertiary structure of DNA: double helix
The DNA chain is 22 to 26 Ångströms wide (2.2 to 2.6 nanometres), and one nucleotide
unit is 3.4 Å (0.34 nm) long.
Sugar-phosphate backbones are not equally-spaced, resulting in major and minor
grooves. One complete turn of the helix requires 3.4 nm (10 bases/turn).
3.6 DNA conformations
DNA exists in several possible conformations. The conformations so far identified are:
A-DNA, B-DNA, C-DNA, D-DNA, E-DNA, H-DNA, L-DNA, and Z-DNA. However, only ADNA, B-DNA, and Z-DNA are believed to be found in nature.
The conformation that DNA adopts depends on the hydration level, DNA sequence, the
amount and direction of supercoiling, chemical modifications of the bases, the type and
concentration of metal ions, as well as the presence of polyamines in solution.
4. DNA replication
DNA replication, the basis for biological inheritance, is a fundamental process occurring
in all living organisms to copy their DNA. This process is "semiconservative" in that each
strand of the original double-stranded DNA molecule serves as template for the reproduction of
the complementary strand. Hence, following DNA replication, two identical DNA molecules
have been produced from a single double-stranded DNA molecule. Cellular proofreading and
error-checking mechanisms ensure near perfect fidelity for DNA replication.
In a cell, DNA replication begins at specific locations in the genome, called "origins".
Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork.
5. DNA self-correction
Imino-cytosine pairs (incorrectly, as far as the cell is concerned) with adenine.
Nanoseconds later, the iC converts back to the normal amino form of cytosine, and no longer
pairs with adenine. The mismatched base sticks out and is cleaved off by the EXO-NUCLEASE
activity of the enzyme, leaving a fresh OH group to try again.
6. DNA damage
DNA can be damaged by many different sorts of mutagens. These include oxidizing
agents, alkylating agents and also high-energy electromagnetic radiation such as ultraviolet light
and x-rays. The type of DNA damage produced depends on the type of mutagen. For example,
UV light mostly damages DNA by producing thymine dimers, which are cross-links between
adjacent pyrimidine bases in a DNA strand. On the other hand, oxidants such as free radicals or
hydrogen peroxide produce multiple forms of damage, including base modifications,
particularly of guanosine, as well as double-strand breaks. It has been estimated that in each
human cell, about 500 bases suffer oxidative damage per day. Of these oxidative lesions, the
most damaging are double-strand breaks, as they can produce point mutations, insertions and
deletions from the DNA sequence, as well as chromosomal translocations.
Many mutagens intercalate into the space between two adjacent base pairs. These
molecules are mostly polycyclic, aromatic, and planar molecules and include ethidium,
proflavin, daunomycin, doxorubicin and thalidomide. DNA intercalators are used in
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chemotherapy to inhibit DNA replication in rapidly-growing cancer cells. In order for an
intercalator to fit between base pairs, the bases must separate, distorting the DNA strand by
unwinding of the double helix. These structural modifications inhibit transcription and
replication processes, causing both toxicity and mutations. As a result, DNA intercalators are
often carcinogens, with benzopyrene diol epoxide, acridines, aflatoxin and ethidium bromide
being well-known examples.
7. DNA reparation
a. PHOTOREACTIVATION (photoenzyme repair) -under the action of the ultra violet
radiation, dimeric bonds appear between adjacent pyrimidin bases, more often between ..T-T..
in one chain which prevents the formation of complementary bonds with A nucleotide from
another chain. Under the action of a visible light photoreactivation with the help of reparative
enzyme takes place. It destroy dimeric bonds and formation of hydrogen bonds between
complementary nucleotides of DNA strands take place (Fig. 2.9).
b. EXCISION REPAIR does not depend on light (dark reparation). In this type of repair
excision of damaged part of one DNA chain by enzyme endonuclease takes place; after that
other enzyme, reparative polymeraze, catalyzes synthesis of a missing part according to the
principle of complementarity and antiparallelism on the remain part of DNA. Then enzyme
ligase joins free ends of a new end with the old ones (Fig. 2.10).
If the damages cannot be removed by the mechanisms mentioned above, they are
removed during recombination between two double strands of DNA. As a result one new
molecule of DNA without damages forms.
8. DNA and RNA comparison. RNA types and properties
Both DNA and RNA are composed of repeating units of nucleotides. Each nucleotide
consists of a sugar, a phosphate and a nucleic acid base.
The sugar in DNA is deoxyribose. The sugar in RNA is ribose, the same as deoxyribose
but with one more OH (oxygen-hydrogen atom combination called a hydroxyl). This is the
biggest difference between DNA and RNA.
Another difference is that RNA molecules can have a much greater variety of nucleic
acid bases. DNA has mostly just 4 different bases with a few extra occasionally. The difference
in these bases (between DNA and RNA) allows RNA molecules to assume a wide variety of
shapes and also many different functions. DNA, on the other hand, serves as a set of directions
and that's about all (but that's absolutely necessary!).
Messanger RNA
- it constitute about 1% in total weight of the cell;
- determines, what amino acids, in what sequence and quantities must be involved in a
polypeptide chain;
- is a primary transcript in prokaryotes;
- is the result of processing in eukaryotes.
Transfer RNA (tRNA)
- the shortest and has a small molecular mass;
- has three levels of spatial arrangement;
- has acceptor and anticodon loops;
- contains the modified nitrogenous bases
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- presence of the modified nitrogenous bases in an anticodon explains the fact of disparity of
amount of codons (61) and tRNA types.
Main functions of rRNA
- structural (makes about 60% ribosome weight). One molecule of rRNA is a part of a small
ribosomal subunit, three molecules build-up a large ribosomal subunit);
- initiation of protein synthesis of (provides interaction of ribosomes with the certain nucleotide
sequences of mRNA and tRNA);
- termination of protein synthesis (determines ending of synthesis and slabbing of the completed
molecules of proteins from ribosomes).
9. Gene definition and classification
A gene is a segment of DNA corresponding to a single protein (or set of alternate protein
variants) or a single (catalytic or structural) RNA molecule.
9.1 Genes’ classification:
Structural genes - genes that code sequence of amino acids of structural proteins and
enzymes,
Genes of modulation - genes that are the cause of other genes activity reduction
(suppressors or inhibitors), the also cause the incretion of other genes activity
(intensificators).
Genes of control (regulation) - genes that render influence to time of another genes
activity. E.g. gene - promoter part goes before the structural genes that respond certain proteins.
Promoter is being recognized by RNA-polymerase thus signaling for m-RNA synthesis to get
started.
Gene - terminator - the end of genes structural part contains a certain sequence of
nucleotides called the terminator. It contains nonsense-triplets. M-RNA synthesis stops here.
RNA-coding genes (rRNA, tRNA), constitutive genes. rRNA genes (4 types) have
information about ribosomal RNA structure and are responsible for their synthesis. tRNA genes
(more than 30 variations) have information about tRNA structure.
9.2 Gene properties
- Discrete action;
-Stability (constancy);
- Liability (change) of genes is connected to their ability to mutations;
- Specificity – every gene is responsible for trait development;
- Pleiotropy - one gene can be responsible for several traits;
- Penetrance and expressivity – frequency of gene viability and level of the trait
expression.
10.Sequences of human genome
It is estimated that only about 5% of the human genome contains actual coding
sequences. Genes for polypeptides include a leader region followed by the coding region
followed by the trailer.
Leader -----> coding region -----> trailer
Intervening sequences separate genes on a strand of DNA.
The leader and trailer are not translated into protein. The coding region is divided into
exons and introns.
Leader -----> coding region -----> trailer
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exons --- introns
While introns are transcribed into immature RNA, they are removed fromwithin the
transcript by splicing together of exons on either side and, thus, do not encode amino acids.
Leader ----(intron---exon---intron---exon---intron)n----trailer
The genes have polarity characterized by a 5’ upstream end and a 3’ downstream end. In
its double stranded form the complementary strands are antiparallel.
10.1 Regulatory Sequences
A class of sequences makes up a numerically insignificant fraction of the genome but
provides critical functions. For example, certain sequences indicate the beginning and end of
genes, sites for initiating replication and recombination, or provide landing sites for proteins
that turn genes on and off. Like structural genes, regulatory sequences are inherited; however,
they are not commonly referred to as genes.
10.2 Repetitive sequences
HIGHLY REPETITIVE CENTROMERIC DNA - tandem repeats in the (untranscribed)
heterochromatin flanking the centromeres are there, but we don't yet know why. One commonly
occurring highly repetitive sequence is the Alu sequence (Alu element) consisting of 300,000 to
500,000 base pairs scattered throughout the human genome. These contain a single site for the
restriction endonuclease AluI.
VNTRs - Variable Number Tandem Repeats are 1-5 kb long, and consist of variable
numbers of adjacent repeats. No one knows what they do, but they are “highly” variable among
individuals. These are the fragments of DNA that are used as DNA FINGERPRINTS, and are
used extensively in criminal forensics. SPACER DNA - Spacer DNA are regions of nontranscribed DNA between tandemly repeated genes, such as ribosomal RNA genes in
eukaryotes. Its function is probably to do with ensuring the high rates of transcription associated
with these genes.
These are sequences that have no product, yet serve a definite function. For example, the
telomere sequences allow replication without reduction in telomere size.
10.3 Gene clusters
A gene cluster is a set of two or more genes that serve to encode for the similar products.
Because populations from a common ancestor tend to possess the same varieties of gene
clusters, they are useful for tracing back recent evolutionary history.
An example of a gene cluster is the Human β-globin gene cluster, which contains five
functional genes and one non-functional gene for similar proteins. Hemoglobin molecules
contain any two identical proteins from this gene cluster, depending on their specific role.
10.4 Pseudogenes
Another class of non-coding DNA is the "pseudogene", so named because it is believed
to be a remnant of a real gene that has suffered mutations and is no longer functional.
Pseudogenes may have arisen through the duplication of a functional gene, followed by
inactivation of one of the copies. Comparing the presence or absence of pseudogenes is one
method used by evolutionary geneticists to group species and to determine relatedness. Thus,
these sequences are thought to carry a record of our evolutionary history.
10.5 Tandemly Repeated (Satellite) DNA
Satellite DNA - DNA of different density: a component of an animal's DNA that differs
in density from surrounding DNA, consists of short repeating sequences of nucleotide pairs,
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and does not undergo transcription
There is remarkable variability in genome size among eukaryotes that has little
correlation with organismal complexity, ploidy or number of coding genes. For example, a
newt has six times the genome size of a human. Much of this variation is due to non-coding,
tandemly repeated DNA. Indeed, a substantial fraction of the genomes of many eukaryotes
is composed of repetitive DNA in which short sequences are tandemly repeated in small to
huge arrays.
Tandemly repetitive sequences, commonly known as "satellite DNAs" are classified
into three major groups:
Satellites are very highly repetitive with repeat lengths of one to several thousand base
pairs. These sequences typically are organized as large (up to 100 million bp !) clusters in the
heterochromatic regions of chromosomes, near centrosomes and telomeres; these are also found
abundantly on the Y chromsome.
Minisatellites are moderately repetitive, tandemly repeated arrays of moderately-sized (9 to
100 bp, but usually about 15 bp) repeats, generally involving mean array lengths of 0.5 to 30
kb. They are found in euchromatic regions of the genome of vertebrates, fungi and plants and
are highly variable in array size.
Microsatellites are moderately repetitive, and composed of arrays of short (2-6 bp) repeats
found in vertebrate, insect and plant genomes. The human genome contains at least 30,000
microsatellite loci located in euchromatin. Copy numbers are characteristically variable within a
population, typically with mean array sizes on the order of 10 to 100. Sometimes is considered
as a VNTR.
Random changes that alter the length of microsatellite DNA near the gene for the
vasopressin receptor affect social behavior in male voles. A longer microsatellite region resulted
in more bonding and care giving.
11.Transposed sequences
Multiple copies of small DNA segments called TRANSPOSABLE GENETIC
ELEMENTS exist throughout the genome, and some can excise and move to different positions
within the genome of a single cell, a process is called transposition.
Their function is not known, and some suspect that they could be remnants of "parasitic"
or "selfish" DNA that simply goes along for the ride without regard for the host.
In the transposition, they can cause mutations and change the amount of DNA in the genome.
Transposons were also once called "jumping genes", and are examples of mobile genetic
elements. They were discovered by Barbara McClintock early in her career, for which she was
awarded a Nobel prize in 1983. She noticed insertions, deletions, and translocations, caused by
these transposons. These changes in the genome could, for example, lead to a change in the
color of corn kernels. About 50% of the total genome of maize consists of transposons.
RETROTRANSPOSONS are similar, but have been reverse transcribed from RNA into DNA
which then inserts into the host genome. These little buggers act a bit like retroviruses.
11.1 Transposons causing diseases
Transposons are mutagens. They can damage the genome of their host cell in different
ways:
A transposon or a retroposon that inserts itself into a functional gene will most likely
disable that gene.
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After a transposon leaves a gene, the resulting gap will probably not be repaired
correctly.
Diseases that are often caused by transposons include hemophilia A and B, severe
combined immunodeficiency, porphyria, predisposition to cancer, and Duchenne muscular
dystrophy.
Additionally, many transposons contain promoters which drive transcription of their own
transposase. These promoters can cause aberrant expression of linked genes, causing disease or
mutant phenotypes.
12. Non-nuclear heredity
A genome is all the genetic information in the haploid portion of chromosomes of a cell.
When the first draft of the human genome sequence became available in February 2001 there
was some surprise that instead of 100,000 genes, only about 30,000 genes were counted. For
example, only about 1.5% of the human genome consists of protein-coding exons, with over
50% of human DNA consisting of non-coding repetitive sequences. The reasons for the
presence of so much non-coding DNA in eukaryotic genomes and the extraordinary differences
in genome size, or C-value, among species represent a long-standing puzzle known as the "Cvalue enigma”. Human Genome researchers have confirmed the existence of 19,599 proteincoding genes in the human genome and identified another 2,188 DNA segments that are
predicted to be protein-coding genes. In general it should be 21,787 protein-coding genes.
Almost a quarter of the protein-coding genes are involved in expression, replication and
maintenance of the genome and another 20% specify components of the signal transduction
pathways that regulate genome expression and other cellular activities in response to signals
received from outside of the cell. Enzymes responsible for the general biochemical functions of
the cell account for another 17.5% of the known genes; the remainder are involved in activities
such as transport of compounds into and out of cells, the folding of proteins into their correct
three-dimensional structures, the immune response, and synthesis of structural proteins such as
those found in the cytoskeleton and in muscles.
12.1 Mitochondrial genome
Not all genetic information is found in nuclear DNA. Both plants and animals have an
organelle—a "little organ" within the cell— called the mitochondrion. Each mitochondrion has
its own set of genes. Mitochondria were once independent living cells similar to today’s
bacteria. Millions of years ago, the bacteria invaded primitive amoeboid cells and established a
mutually beneficial (symbiotic) relationship. So, human cells probably evolved as symbiotic
cellular communities. Over millions of years, redundant genes were lost from the bacteria and
they became entirely dependent on their hosts, ceasing to exist as independent life forms. Cells
often have multiple mitochondria, particularly cells requiring lots of energy, such as active
muscle cells. Unlike nuclear DNA (the DNA found within the nucleus of a cell), half of which
comes from our mother and half from our father, mitochondrial DNA is only inherited from our
mother. This is because mitochondria are only found in the female gametes or "eggs" of
sexually reproducing animals, not in the male gamete, or sperm. Mitochondrial DNA also does
not recombine; there is no shuffling of genes from one generation to the other, as there is with
nuclear genes. Large numbers of mitochondria are found in the tail of sperm, providing them
with an engine that generates the energy needed for swimming toward the egg. However, when
the sperm enters the egg during fertilization, the tail falls off, taking away the father's
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mitochondria.
Human mitochondrial genome is a small circular DNA molecule 16 568 bp in length
containing 37 genes.
Twenty-four of mitochondrial genes specify RNA molecules involved in protein
synthesis while the remaining 13 encode proteins required for the biochemical reactions that
make up respiration (ATP synthesis).
A number of rare diseases are caused by mutations in mitochondrial DNA, and the tissues
primarily affected are those that most rely on respiration, i.e. the brain and nervous system,
muscles, and the kidneys and liver.
Mitochondrial diseases include Leber's hereditary optic neuropathy, in which there is loss
of vision often combined with cardiac arrhythmia, and Kearns-Sayre syndrome, which involves
paralysis of the eye muscles, dementia and seizures.
LECTURE 4
Of Special Course “Modern Problems of Molecular Biology”
for Pharmaceutical Department students of the 2nd Year of Study
THEME:
“MOLECULAR MECHANISMS OF GENE, CHROMOSOMAL AND GENOMIC
MUTATIONS”
Lecture plan:
1. Mutation definition. Mutant bodies
2. Mutation origin and inheritance
3. Mutation effect on an organism
4. Beneficial mutations
5. Harmful mutations
6. Antimutagenic effect
7. Mutation types
7.1 Gene mutations
7.2 Chromosomal abberations
7.3 Genomic mutations
8. Mosaicim
9. Copy number variation
10. Frequency of mutations
1. Mutation definition. Mutant bodies
Mutation is defined as a permanent transmissible change in a DNA sequence away from
normal that has not been repaired.
Organisms in which mutations took place are named mutants.
2. Mutation origin and inheritance
Mutations occur naturally, caused by errors in DNA duplication, errors in processing
DNA and errors in meiosis and mitosis. Physical damage and chemical damage can induce
mutations as well, and are used by researchers to study mutations.
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If the frequency of the gene’ change is lower that 1per cent in the population, the allele is
regarded as a mutation. This implies there is a normal allele that is prevalent in the population
and that the mutation changes this to a rare and abnormal variant. Mutations range in size from
a single DNA building block (DNA base) to a large segment of a chromosome.
Mutations occur in two ways: they can be inherited from a parent or acquired during a
person’s lifetime.
Mutations that are passed from parent to child are called hereditary mutations or germline
mutations (because they are present in the egg and sperm cells, which are also called germ
cells). This type of mutation is present throughout a person’s life in virtually every cell in the
body.
Mutations that occur only in an egg or sperm cell, or those that occur just after fertilization, are
called new (de novo) mutations. De novo mutations may explain genetic disorders in which an
affected child has a mutation in every cell, but has no family history of the disorder.
Acquired (or somatic) mutations occur in the DNA of individual cells at some time during a
person’s life. These changes can be caused by environmental factors such as ultraviolet
radiation from the sun, or can occur if a mistake is made as DNA copies itself during cell
division. Acquired mutations in somatic cells cannot be passed on to the next generation.
Some genetic changes are very rare; others are common in the population. Genetic changes that
occur in more than 1 percent of the population are called polymorphisms. They are common
enough to be considered a normal variation in the DNA. Polymorphisms are responsible for
many of the normal differences between people such as eye color, hair color, and blood type.
Although many polymorphisms have no negative effects on a person’s health, some of these
variations may influence the risk of developing certain disorders.
2. Mutation effect on an organism
The characteristics of organisms are determined by their genetic material (DNA), and
random mutations (changes) in the DNA can result in slight changes in organisms. As these
accumulate, there can be changes in organisms, resulting in evolution.
About 90 percent of DNA is thought to be non-functional, and mutations there generally have
no effect. The remaining 10 percent is functional, and has an influence on the properties of an
organism, as it is used to direct the synthesis of proteins that guide the metabolism of the
organism. Mutations to this 10 percent can be neutral, beneficial, or harmful. Probably less than
half of the mutations to this 10 percent of DNA are neutral.
Harmful mutations result in organisms less likely to survive, and so these mutations tend
to be eliminated from the population (group of organisms in a species).
Beneficial mutations also tend to be eliminated by chance, but less often, and tend to be
preserved. As these accumulate, the species can gradually adapt to its environment. Neutral
mutations are generally eliminated, curiously, but sometimes can spread to the whole
population. We then say that the mutation has fixed in the population. The rate of evolution is
the rate at which mutations fix in the population.
4. Beneficial mutations
Although most mutations that change protein sequences are harmful, some mutations
have a positive effect on an organism. In this case, the mutation may enable the mutant
organism to withstand particular environmental stresses better than wild-type organisms, or
reproduce more quickly. In these cases a mutation will tend to become more common in a
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population through natural selection.
For example, a specific 32 base pair deletion in human CCR5 (CCR5-Δ32) confers HIV
resistance to homozygotes and delays AIDS onset in heterozygotes. The CCR5 mutation is
more common in those of European descent. One possible explanation of the etiology of the
relatively high frequency of CCR5-Δ32 in the European population is that it conferred
resistance to the bubonic plague in mid-14th century Europe. People with this mutation were
more likely to survive infection; thus its frequency in the population increased. This theory
could explain why this mutation is not found in Africa, where the bubonic plague never
reached. A newer theory suggests that the selective pressure on the CCR5 Delta 32 mutation
was caused by smallpox instead of the bubonic plague.
5. Harmful mutations
Changes in DNA caused by mutation can cause errors in protein sequence, creating
partially or completely non-functional proteins. To function correctly, each cell depends on
thousands of proteins to function in the right places at the right times. When a mutation alters a
protein that plays a critical role in the body, a medical condition can result. A condition caused
by mutations in one or more genes is called a genetic disorder. Some mutations alter a gene's
DNA base sequence but do not change the function of the protein made by the gene. Studies of
the fly Drosophila melanogaster suggest that if a mutation does change a protein, this will
probably be harmful, with about 70 percent of these mutations having damaging effects, and the
remainder being either neutral or weakly beneficial. However, studies in yeast have shown that
only 7% of mutations that are not in genes are harmful.
If a mutation is present in a germ cell, it can give rise to offspring that carries the
mutation in all of its cells. This is the case in hereditary diseases. On the other hand, a mutation
may occur in a somatic cell of an organism. Such mutations will be present in all descendants of
this cell within the same organism, and certain mutations can cause the cell to become
malignant, and thus cause cancer.
Often, gene mutations that could cause a genetic disorder are repaired by the DNA repair
system of the cell. Each cell has a number of pathways through which enzymes recognize and
repair mistakes in DNA. Because DNA can be damaged or mutated in many ways, the process
of DNA repair is an important way in which the body protects itself from disease.
6. Antimutagenic effect
The nature had created several protective mechanisms during the evolution which
decrease frequency of phenotypic manifestations of mutations.
I. Degeneracy of biological code, i.e. one amino acid is specified by several triplets.
II. Mutant genes are often recessive and do not manifest themselves at heterozygous state
because of the diploid set of chromosomes (normal allele gene).
III. Reparation of DNA structure damaged by the action of mutagen, repetitions of genes,
double chain of DNA, and homologous pairs of chromosomes. The reparations can occurs in
two ways:
Photoreactivation (photoenzyme repair, see Chapter 2) -under the action of the ultraviolet
radiation reparation of dimeric bonds appear between adjacent pyrimidin bases, more often
between T and T in one chain occurs. T-T links prevents the formation of complementary bonds
with A nucleotide from another chain.
Excision repair does not depend on light (dark reparation). In this type of repair excision
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of damaged part of one DNA chain by enzyme endonuclease takes place.
7. Mutation types
7.1 Gene mutations
Gene mutations are characterized by changes of the nucleotides normal sequence. Normal
gene and mutant one formed from it are allelic. There are the following types of gene mutations:
Replacement of nucleotides - one nucleotide appears instead of another. There are 2
types of replacement:
Missense mutation - This type of mutation is a change in one DNA base pair that results
in the substitution of one amino acid for another in the protein made by a gene.
Nonsense mutation - A nonsense mutation is also a change in one DNA base pair. Instead
of substituting one amino acid for another, however, the altered DNA sequence prematurely
signals the cell to stop building a protein. This type of mutation results in a shortened protein
that may function improperly or not at all.
These mutations appear constantly and are the most frequent. They have an important
meaning for evolution of nature and for selection of practically important mutants.
The sickle-cell anemia is an example of mutation caused by the nucleotides replacement.
Under this disease amino acid glutamine is replaced by valine in beta-chain of hemoglobin in
sixth position. TTG appears instead of glutamine-coding triplet GAG.
Gene mutations may either manifest in change of a character or not, so mutations can't be
revealed.
A degree of character change depends on polypeptide chain structure change and protein
function, e.g. enzyme. It should be taken into account that mutation appear only in one of allele
gene and its manifestation depends on interaction with a normal allele in heterozygotes.
Insertion - An insertion changes the number of DNA bases in a gene by adding a piece of
DNA. As a result, the protein made by the gene may not function properly.
Duplication - A duplication consists of a piece of DNA that is abnormally copied one or
more times. This type of mutation may alter the function of the resulting protein.
Repeat expansion - Nucleotide repeats are short DNA sequences that are repeated a
number of times in a row. For example, a trinucleotide repeat is made up of 3-base-pair
sequences, and a tetranucleotide repeat is made up of 4-base-pair sequences. A repeat expansion
is a mutation that increases the number of times that the short DNA sequence is repeated. This
type of mutation can cause the resulting protein to function improperly.
7.2 Chromosomal abberations
Duplications
A deleted chromosome fragment can attach to its homologue, thereby duplicating a
region of genes on the chromosome to which it attaches. Or, as happens in some genes, a
segment of the gene or chromosome undergoes multiple repetitions, so that several copies are
located on the chromosome. A common duplication, the trinucleotide repeat, occurs within
some abnormal genes, and is responsible for several genetic disorders, including fragile X
syndrome and Huntington's disease. In vertebrates, hemoglobin genes may have evolved by
duplication. Hemoglobin is composed of alpha and beta sub-units, which are coded by genes
that have a similarity of 75%, suggesting a common origin by duplication followed by
divergence.
Example - Fragile X: the most common form of mental retardation. The X chromosome
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of some people is unusually fragile at one tip - seen "hanging by a thread" under a microscope.
Most people have 29 "repeats" at this end of their X-chromosome, those with Fragile X have
over 700 repeats due to duplications. Affects in USA 1:1500 males, 1:2500 females.
Chromosomal translocation
A gene can be transposed or translocated (moved) to a different location along the
chromosome, so that the sequence might read A-B-C-E-F-G-D rather than A-B-C-D-E-F-G.
Reciprocal translocations involve exchange of genes between non-homologous
chromosomes. The translocation of a piece of the human 22 chromosome to the 9 chromosome
causes Acute Myelogenous Leukemia because it interferes with a gene that controls cell
division. This abnormal chromosome is called the Philadelphia chromosome, from the city in
which the researchers who discovered this abnormality lived.Fusion of the long arms of two
acrocentric chromosomes [13,14,15,21,22] into a single chromosome having lost the short arms
at the same time. Most often occurs as 21/21, 13/14, and 14/21 translocations. Apart from being
an important cause of uniparental disomy, it may cause trisomy 21 (Down's syndrome) in the
offspring. Human chromosome 2 is a result of a centric fusion between two ancestral ape
chromosomes (gorillas have 24 pairs of chromosomes).
Ring chromosomes usually occur when a chromosome breaks in two places and the ends of the
chromosome arms fuse together to form a circular structure. The ring may or may not
include the chromosome’s constriction point (centromere). In many cases, genetic material
near the ends of the chromosome is lost.
Dicentric chromosomes - unlike normal chromosomes, which have a single constriction point
(centromere), a dicentric chromosome contains two centromeres. Dicentric chromosomes
result from the abnormal fusion of two chromosome pieces, each of which includes a
centromere. These structures are unstable and often involve a loss of some genetic
material.
7.3 Genomic mutations
involves the changing of chromosomes number of whole chromosome sets. A set of
chromosomes in sex cell (gamete) is called genome. Genomic mutations are subdivided into: 1)
polyploidy — the state of having more than diploid set of chromosomes (3n—triploid, 4n—
tetraploid, 5n — pentaploid, 6n—hexaploid). Many plant species are polyploid.
Aneuploidy - a genetic change that involves the loss or gain of entire chromosomes. Due
to problems in the cell division process, the replicated chromosomes may not separate into the
daughter cells accurately. This can result in cells that have too many chromosomes or too few
chromosomes. An example of a fairly common aneuploid condition that is unrelated to cancer is
Down syndrome, in which there is an extra copy of chromosome 21 in all of the cells of the
affected individual.
The most common types of aneuploids are:
1. Nullisomics: A whole pair of chromosomes is missing, expressed as 2n-2.
2. Monosomics: One homologue is missing, expressed as 2n-1.
3. Trisomics: Individuals with and extra chromosome. One pair has three instead of two
chromosomes, expressed as 2n+1.
8. Mosaicism
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Mutations may also occur in a single cell within an early embryo. As all the cells divide
during growth and development, the individual will have some cells with the mutation and
some cells without the genetic change. This situation is called mosaicism.
9. Copy number variation
In some cases the number of copies varies—meaning that a person can be born with one,
three, or more copies of particular genes. Less commonly, one or more genes may be entirely
missing. This type of genetic difference is known as copy number variation (CNV).
Copy number variation results from insertions, deletions, and duplications of large
segments of DNA. These segments are big enough to include whole genes. Variation in gene
copy number can influence the activity of genes and ultimately affect many body functions.
Researchers were surprised to learn that copy number variation accounts for a significant
amount of genetic difference between people. More than 10 percent of human DNA appears to
contain these differences in gene copy number. While much of this variation does not affect
health or development, some differences likely influence a person’s risk of disease and response
to certain drugs. Future research will focus on the consequences of copy number variation in
different parts of the genome and study the contribution of these variations to many types of
disease.
10.Frequency of mutations
Both genetic and non-genetic factors influence frequency of mutations
Properties of a given locus. It has been calculated that an average frequency of mutations
is from 10-5 to 10-6. But genes of one type are more mutable and the other are more stable. E.g.,
in a human being gene of haemophilia A mutates with the frequency of 30 - 50 x 10-6 and that
of haemophilia B with frequency of 2-3 x 10-6.
The properties of an organism where a gene functions. It is established that mutations by
hemophilia B in women are met 10 times more frequent than in men.
Factors on non-genetic nature influence the frequency of mutations. It is supposed that
their number enlarges in a human being with age, influenced by ultraviolet rays etc.
The process of genetic material change is called mutagenesis (G.de Freeze). There are
spontaneous and induced mutagenesis.
Spontaneous mutagenesis (SM) is a name for changes of inherited material under natural
conditions without visible reasons. Though they exist and the main mechanisms are:
-errors of DNA duplication lead to the replacement of bases in nucleotides;
-errors of DNA reparation fixing the changes had happened;
- presence of special genes - mutators, inducing mutagenesis;
- ability of separate genes to migrate;
- accumulation of definite chemical substances in the cells during which later on damages
genie apparatus.
Induced mutagenesis is an artificial increase of mutations frequincy under influence of
non-physiologic doses of mutagenes. Mutagenes are substances causing mutations. They are
subdivided into physical, chemical, biological. Among physical mutagenes ionizing radiation is
on the first place. It's mutagenic influence on a human being is increased because of wide use of
radioactive substances in industry, X-ray diagnosis, medicine. Consequences of Chernobyl
accident are of great importance in this respect.
The spectrum of chemical mutagenes is extremely wide. These are chemical substances
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of harmful chemical manufactures, different chemical substances used in an agriculture, the
ones used in domestic needs, Pharmaceuticals.
Biological mutagenes are viruses that able to insert into a human being genome and
damage.
LECTURE 5
Of Special Course “Modern Problems of Molecular Biology”
for Pharmaceutical Department students of the 2nd Year of Study
THEME:
“DNA METHODS. RECOMBINANT DNA”
Lecture Plan:
1. Recombinant DNA technology definition
2. Direct gene testing
3. DNA methods
4. Sources of the genetic material for DNA testing
5. Step 1: DNA Restriction
5.1Creation of the recombinant DNA
6. Polymerase chain reaction (PCR)
7. Step 2: Gel electrophoresis
8. Step 3: DNA hybridization
9. Southern blot
10.Indirect gene tracking (linkage)
10.1
Restriction fragment length polymorphism
10.2
DNA chips
10.3
DNA profiling
11.Predictive Genetic Testing
12.DNA sequencing
13.The Western blot
14.The Northern blot
15.Eastern blotting
16.Southwestern blotting
17.Practical Applications of DNA Technology
18.Detection of a Gene
1. Recombinant DNA technology definition
a collection of experimental techniques, which allow for isolation, copying & insertion of
new DNA sequences into host-recipient cells by a number of laboratory protocols &
methodologies.
2. Direct gene testing
looks at the presence or absence of a known gene mutation by examining the sequence of
nucleotides in the information in the gene
The test is very accurate and used for diagnosis and screening including prenatal, genetic
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carrier testing and screening, presymptomatic and predictive testing
Limitations include:
– Interpretation of the test result eg. finding that a person has a faulty gene does not
always relate to how a person is, or will be, affected by that condition
– The testing may be time-consuming and expensive for the health service if not for the
patient
– For some complex conditions eg. cancer, the testing may have to be done on a family
member with the condition to identify a family-specific mutation in the gene (mutation
searching) before unaffected family members can be offered predictive testing
3. DNA methods:
DNA sequencing - это узнавание последовательности оснований ДНК
DNA cloning - - размножение отдельных фрагментов ДНК
Reverse transcription - получение определенных фрагментов ДНК (зондов ДНК) на основе
обратной транскрипции с мРНК
DNA hybridization - путем генной инженерии
Polymerase chain reaction (PCR)
FISH - analysis
4. Sources of the genetic material for DNA testing
DNA to be tested can be extracted from the cells of a variety of body fluids or tissues.
While the majority of tests are carried out using DNA from blood cells (lymphocytes), cells
obtained from the lining of the cheek using a mouth-wash or the cells in the roots of an
individual’s hair may also be sources of DNA.
5. Step 1: DNA Restriction
In the 40 - 50 years that molecular biology has existed, scientists have used restriction
mapping to analyze the structure and sequence of DNA molecules. This powerful technique
involves the use of restriction digestion.
Restriction Digestion is the process of cutting DNA molecules into smaller pieces with
special enzymes called Restriction Endonucleases (sometimes just called Restriction Enzymes
or RE).
REs recognize specific DNA sequences wherever that sequence occurs in the DNA,
usually 4 to 8 bp in length (for example GATATC), and cleave at these sequences.
As everyone’s DNA has some small differences, the sites may be at different places in
people’s non-coding DNA and so the enzymes will cut the DNA into different sizes in different
people.
Nathans, Smith and Arber were awarded the Nobel Prize in 1979 for discovering
restriction enzymes and having the insight and creativity to use these enzymes to map genes.
More than 900 restriction enzymes, some sequence specific and some not, have been isolated
from over 230 strains of bacteria since.
Restriction enzymes and the fragments produced by them have become powerful tools of
molecular genetics. They are used
to map DNA molecules physically,
to analyze population polymorphisms,
to rearrange DNA molecules,
to prepare molecular probes,
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to create mutants,
to analyze the modification status of the DNA, and other applications.
There are three classes of restriction enzymes (type I, II and III). The most commonly
used ones are type II enzymes. These recognize specific sequences that are 4, 5, and 6
nucleotides in length and display a twofold symmetry. Recognition sequences for many type II
enzymes are the same on both strands. Such recognition sequences are said to be palindromic.
Some RE cleave both strands exactly at the axis of symmetry, generating fragments of DNA
that carry blunt or flushed ends (e.g. EcoRV); others cleave each strand at similar locations on
opposite sides of the axis of symmetry, creating fragments of DNA that carry protruding singlestranded termini (also called “sticky” ends or overhangs).
5.1Creation of the recombinant DNA
Restriction Endonucleases - diplotomic cuts at unique DNA sequences, mostly
palindromes.
DNA's cut this way have sticky (complimentary) ends & can be reannealed or spliced
with other DNA molecules to produce new genes combinations and sealed via DNA ligase.
6. Polymerase chain reaction (PCR)
In molecular biology, the PCR is a technique to amplify a single or few copies of a piece
of DNA across several orders of magnitude, generating thousands to millions of copies of a
particular DNA sequence. The method relies on thermal cycling, consisting of cycles of
repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the
DNA. Primers (short DNA fragments) containing sequences complementary to the target region
along with a DNA polymerase (after which the method is named) are key components to enable
selective and repeated amplification.
As PCR progresses, the DNA generated is itself used as a template for replication, setting
in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be
extensively modified to perform a wide array of genetic manipulations.
Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq
polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA
polymerase enzymatically assembles a new DNA strand from DNA building blocks, the
nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called
DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR
methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined
series of temperature steps. These thermal cycling steps are necessary first to physically
separate the two strands in a DNA double helix at a high temperature in a process called DNA
melting. At a lower temperature, each strand is then used as the template in DNA synthesis by
the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from
the use of primers that are complementary to the DNA region targeted for amplification under
specific thermal cycling conditions.
6.Polymerase chain reaction (PCR)
Developed in 1984 by Kary Mullis, PCR is now a common and often indispensable
technique used in medical and biological research labs for a variety of applications. These
include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes;
the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic
20
sciences and paternity testing); and the detection and diagnosis of infectious diseases. In 1993
Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.
7. Step 2: Gel electrophoresis
Gel electrophoresis is a technique used for the separation of nucleic acids and proteins.
The cut DNA is placed into a slab of ‘jelly’ (a gel matrix) and an electrical current is applied so
that the ‘jelly’ becomes electrified and has a ‘positive’ (+) end at the top and a negative (-) end
at the bottom - just like the positive and negative ends of a battery.
As the DNA is a chemical which has a negative charge, the DNA moves towards the
positive end of the gel or from the top to the bottom.
The pieces of DNA separate on the gel according to size: the biggest pieces move the
slowest and so will be closest to the top of the gel. The gel now contains all of the individual’s
DNA spread from the top to the bottom of the gel.
The frictional force of the gel material acts as a "molecular sieve," separating the
molecules by size. During electrophoresis, macromolecules are forced to move through the
pores when the electrical current is applied. Their rate of migration through the electric field
depends on the strength of the field, size and shape of the molecules, relative hydrophobicity of
the samples, and on the ionic strength and temperature of the buffer in which the molecules are
moving. After staining, the separated macromolecules in each lane can be seen in a series of
bands spread from one end of the gel to the other.
8. Step 3: DNA hybridization
To select out the pieces of DNA that need to be analysed, the pieces of DNA that have
spread through the gel are covered with special DNA ‘probes’. The probes have been made in
the laboratory and contain a match for the DNA sequence that the test is designed to identify.
The probes in fact have the opposite letters in the genetic code sequence to the sequence in the
gene or DNA segment that needs to be isolated. The two sequences match up because of the
ability of the letters A and T, and C and G to pair with each other as shown in Figure.
The development of the probes used is critical. They can be expensive to develop and the
process may take some time.
Recent developments have enabled faster testing to see if a genetic condition is due to
having the loss of copies of particular gene(s) (deletion) or too many copies (duplication). The
probes are produced in the form of microarrays.
The same principles described above are used in microarray testing except that the DNA
from the person being tested is applied to a very small unit on which thousands of different
‘probes’ representing thousands of regions of DNA or genes have been placed. Microarrays can
be built that are specific for one particular chromosome or include all of the DNA in a human
cell (the genome). An example of the result of a DNA genetic test as seen in the laboratory is
shown in Figure. There are two copies of each gene. In this case
Person A has two faulty copies of a gene and may have the genetic condition
Person B has one copy that is faulty and the other is working.
Therefore this person is a carrier of the faulty gene
Person C has both copies of this gene containing the right information and has normal
gene function. The DNA examination may involve the analysis of the gene itself (direct gene
testing) or of short segments of the DNA close to or within a gene (indirect gene tracking or
linkage).
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9. Southern blot
is a method routinely used in molecular biology for detection of a specific DNA sequence
in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA
fragments to a filter membrane and subsequent fragment detection by probe hybridization. The
method is named after its inventor, the British biologist Edwin Southern. Other blotting
methods (i.e., western blot, northern blot, eastern blot, southwestern blot) that employ similar
principles, but using RNA or protein, have later been named in reference to Edwin Southern's
name. As the technique was eponymously named, Southern blot should be capitalized as is
required for proper nouns, whereas names for other blotting methods should not.
Hybridization of the probe to a specific DNA fragment on the filter membrane indicates
that this fragment contains DNA sequence that is complementary to the probe.
The transfer step of the DNA from the electrophoresis gel to a membrane permits easy
binding of the labeled hybridization probe to the size-fractionated DNA. It also allows for the
fixation of the target-probe hybrids, required for analysis by autoradiography or other detection
methods.
Southern blots performed with restriction enzyme-digested genomic DNA may be used to
determine the number of sequences (e.g., gene copies) in a genome. A probe that hybridizes
only to a single DNA segment that has not been cut by the restriction enzyme will produce a
single band on a Southern blot, whereas multiple bands will likely be observed when the probe
hybridizes to several highly similar sequences (e.g., those that may be the result of sequence
duplication). Modification of the hybridization conditions (for example, increasing the
hybridization temperature or decreasing salt concentration) may be used to increase specificity
and decrease hybridization of the probe to sequences that are less than 100% similar.
10.Indirect gene tracking (linkage)
relies on comparing DNA markers from family members with the condition to markers in
unaffected relatives. Used in situations where the gene itself has not been precisely located or
where mutation(s) in a gene have not yet been defined; the test is not as accurate as direct gene
testing but can be used in diagnosis including prenatal and presymptomatic and predictive
testing.
Limitations include: It may not always be possible to find DNA markers that enable the
scientists to tell the difference between the faulty gene copy and the working gene copy.
10.1 Restriction fragment length polymorphism
The term restriction fragment length polymorphism, or RFLP, (commonly
pronounced “rif-lip”) refers to a difference between two or more samples of homologous DNA
molecules arising from differing locations of restriction sites, and to a related laboratory
technique by which these segments can be distinguished. In RFLP analysis the DNA sample is
broken into pieces (digested) by restriction enzymes and the resulting restriction fragments are
separated according to their lengths by gel electrophoresis and transferred to a membrane via
the Southern blot procedure. Hybridization of the membrane to a labeled DNA probe then
determines the length of the fragments which are complementary to the probe. A RFLP occurs
when the length of a detected fragment varies between individuals. Each fragment length is
considered an allele, and can be used in genetic analysis.
Although now largely obsolete, RFLP analysis was the first DNA profiling technique
inexpensive enough to see widespread application. In addition to genetic fingerprinting, RFLP
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was an important tool in genome mapping, localization of genes for genetic disorders,
determination of risk for disease, and paternity testing. Most RFLP markers are co-dominant
(both alleles in heterozygous sample will be detected) and highly locus-specific.
RFLP analysis may be subdivided into single- (SLP) and multi-locus probe (MLP)
paradigms. Usually, the SLP method is preferred over MLP because it is more sensitive, easier
to interpret and capable of analyzing mixed-DNA samples. Moreover data can be generated
even when the DNA is degraded (e.g. when it is found in bone remains.)
In this method of genetic testing, scientists use the fact that there are special segments of
DNA that are located very close to the gene on the same chromosome. These segments nearly
always travel with the gene when it is passed from parent to child: this is more likely the closer
they are to the gene.
These segments of DNA are called ‘polymorphic markers’: “poly” means many and
morphic means forms. These markers are different in different families. They are a bit like
flashing lights that warn them that either the working copy of the gene, or the faulty copy
containing the mutation, is nearby.
The closer the markers are linked to the gene, the more confident the scientist can be that
a marker is travelling with either the working copy or the faulty gene copy. This method of
indirect gene tracking is referred to as the linkage method.
The markers that are linked to the faulty or working gene copies are special to each
family, so this method of genetic testing can only be done within families. Indirect gene
tracking is a ‘family test’.
10.2 DNA chips
can reveal DNA mutations and RNA expression. There are a large number of genes in
eukaryotic genomes.
The pattern of expression between different tissues and at different times is quite
distinctive.
Cells have unique mRNA's.
For example, early stage skin cancer have a unique mRNA "fingerprint".
To find these patterns, DNA sequences can be arranged in an array on a solid support.
DNA chip technology provides these large arrays.
Merging DNA technology with the manufacturing technology of the semiconductor
industry, large arrays are being produced.
DNA chips are glass slides onto which DNA sequences are attached in precise order.
The typical slide is divided into 24x24 uM squares. Each contains about 10 million copies
of a particular sequence, which is up to 20 nucleotides long.
A computer controls the additions of the nucleotides in predetermined pattern.
Up to 60,000 different sequences can be put on a single chip.
Cellular mRNA is isolated from cells and is used to make complementary DNA, which is
called cDNA.
Reverse transcriptase and PCR are used together in a process called RT-PCR.
The amplified cDNA is coupled to a fluorescent dye. It is then hybridized to the chip.
A sensitive scanner detects the spots on the array that glow. The combinations of spots
that light up differ with different types of cells or different physiological states.
DNA chip technology can be used in detecting genetic variants.
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Twenty-nucleotide fragments of DNA sequences of all possible mutations are arranged.
The person's DNA is hybridized to determine if any hybridize to a mutant sequence on
the chip.
10.3 DNA profiling
(also called DNA testing, DNA typing, or genetic fingerprinting)
— is a technique employed by forensic scientists to assist in the identification of
individuals on the basis of their respective DNA profiles. DNA profiles are encrypted sets of
numbers that reflect a person's DNA makeup, which can also be used as the person's identifier.
DNA profiling should not be confused with full genome sequencing. It is used in, for example,
parental testing and rape investigation.
Although 99.9% of human DNA sequences are the same in every person, enough of the
DNA is different to distinguish one individual from another. DNA profiling uses repetitive
("repeat") sequences that are highly variable, called variable number tandem repeats (VNTR).
VNTRs loci are very similar between closely related humans, but so variable that unrelated
individuals are extremely unlikely to have the same VNTRs.
The DNA profiling technique was first reported in 1984 by Sir Alec Jeffreys at the
University of Leicester in England, and is now the basis of several national DNA databases.
11.Predictive Genetic Testing
Sometimes the detection of the faulty gene provides the person with an increased risk
estimate, rather than certainty, that they will develop a particular condition later in life. This
type of direct gene testing is called predictive testing.
Predictive testing for some families is available for inherited conditions such as an
inherited predisposition to haemochromatosis or breast cancer.
12.DNA sequencing
Once a gene has been located precisely on a chromosome, the next steps are for scientists
to determine the normal chemical structure of the gene and the changes that alter the coded
message.
Maxam-Gilbert sequencing
In 1976–1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method
based on chemical modification of DNA and subsequent cleavage at specific bases. Although
Maxam and Gilbert published their chemical sequencing method two years after the groundbreaking paper of Sanger and Coulson on plus-minus sequencing, Maxam-Gilbert sequencing
rapidly became more popular, since purified DNA could be used directly, while the initial
Sanger method required that each read start be cloned for production of single-stranded DNA.
However, with the improvement of the chain-termination method (see below), Maxam-Gilbert
sequencing has fallen out of favour due to its technical complexity prohibiting its use in
standard molecular biology kits, extensive use of hazardous chemicals, and difficulties with
scale-up.
The method requires radioactive labelling at one end and purification of the DNA
fragment to be sequenced. Chemical treatment generates breaks at a small proportion of one or
two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of
labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each
molecule. The fragments in the four reactions are arranged side by side in gel electrophoresis
for size separation. To visualize the fragments, the gel is exposed to X-ray fil for
24
autoradiography, yielding a series of dark bands each corresponding to a radiolabelled DNA
fragment, from which the sequence may be inferred.
Also sometimes known as "chemical sequencing", this method originated in the study of
DNA-protein interactions (footprinting), nucleic acid structure and epigenetic modifications to
DNA, and within these it still has important applications.
13.The Western blot
(alternatively, protein immunoblot) is an analytical technique used to detect specific
proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to
separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or
by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then
transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected)
using antibodies specific to the target protein.
There are now many reagent companies that specialize in providing antibodies (both
monoclonal and polyclonal antibodies) against tens of thousands of different proteins.
Commercial antibodies can be expensive, although the unbound antibody can be reused
between experiments. This method is used in the fields of molecular biology, biochemistry,
immunogenetics and other molecular biology disciplines.
Other related techniques include using antibodies to detect proteins in tissues and cells by
immunostaining and enzyme-linked immunosorbent assay (ELISA).
The method originated from the laboratory of George Stark at Stanford. The name
western blot was given to the technique by W. Neal Burnette and is a play on the name
Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection
of RNA is termed northern blotting and the detection of post-translational modification of
protein is termed Eastern blotting.
14.The Northern blot
is a technique used in molecular biology research to study gene expression by detection
of RNA (or isolated mRNA) in a sample.
With northern blotting it is possible to observe cellular control over structure and function
by determining the particular gene expression levels during differentiation, morphogenesis, as
well as abnormal or diseased conditions. Northern blotting involves the use of electrophoresis to
separate RNA samples by size, and detection with a hybridization probe complementary to part
of or the entire target sequence. The term 'northern blot' actually refers specifically to the
capillary transfer of RNA from the electrophoresis gel to the blotting membrane, however the
entire process is commonly referred to as northern blotting.
The northern blot technique was developed in 1977 by James Alwine, David Kemp, and
George Stark at Stanford University. Northern blotting takes its name from its similarity to the
first blotting technique, the Southern blot, named for biologist Edwin Southern.
15.Eastern blotting
is a technique to analyze proteins, lipids, or glycoconjugates, and is most often used to
detect carbohydrate epitopes. Thus, Eastern blotting can be considered an extension of the
biochemical technique of western blotting which detects protein post translational modifications
(PTM). Multiple techniques have been described by the term Eastern blotting, most use proteins
or lipids blotted from SDS-PAGE gel on to a PVDF or nitrocellulose membrane. Transferred
proteins are analyzed for post-translational modifications using probes that may detect lipids,
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carbohydrate, phosphorylation or any other protein modification. Eastern blotting should be
used to refer to methods that detect their targets through specific interaction of the PTM and the
probe, distinguishing them from a standard Far-western blot. In principle, Eastern blotting is
similar to lectin blotting (i.e. detection of carbohydrate epitopes on proteins or lipids); however,
the term lectin blotting is more prevalent in the literature.
16.Southwestern blotting
based along the lines of Southern blotting (which was created by Edwin Southern) and
first described by B. Bowen and colleagues in 1980, is a lab technique which involves
identifying and characterizing DNA-binding proteins (proteins that bind to DNA) by their
ability to bind to specific oligonucleotide probes. The proteins undergo gel electrophoresis and
are subsequently transferred to nitrocellulose membranes similar to other types of blotting.
The name southwestern blotting is based on the fact that this technique detects DNAbinding proteins, since DNA detection is by Southern blotting and protein detection is by
western blotting.
However, since the first southwestern blottings, many more have been proposed and
discovered. Large amounts of proteins and their degradation when being isolated hampered
previous protocols.
"Southwestern blot mapping" is performed for rapid characterization of both DNAbinding proteins and their specific sites on genomic DNA. Proteins are separated on a Sodium
Dodecyl Sulfate (SDS), polyacrylamide gel (PAGE), renatured by removing SDS in the
presence of urea, and blotted onto nitrocellulose by diffusion. The genomic DNA region of
interest is digested by restriction enzymes selected to produce fragments of appropriate but
different sizes, which are subsequently end-labeled and allowed to bind to the separated
proteins. The specifically bound DNA is eluted from each individual protein-DNA complex and
analyzed by acrylamide gel electrophoresis. Evidence that tissue-specific DNA binding proteins
may be detected by this technique is presented. Moreover, their sequence-specific binding
allows the purification of the corresponding selectively bound DNA fragments and may
improve protein-mediated cloning of DNA regulatory sequences.
17. Practical Applications of DNA Technology
1. Medical: disease often involves changes in gene expression, so DNA methods
- help to found failed gene.
- A disease/infection diagnosis is also possible: PCR & labeled DNA probes from
pathogens can help identify microbe types by Restriction Fragment Length Analysis (RFLP)
when markers often inherited with disease.
- Fragment analysis (DNA fingerprinting) also used for paternity testing
- Gene Therapy: idea is to replace defective genes via microinjection of DNA requires
vectors.
2. Pharmaceutical Products: manufactured drugs.
18. Detection of a Gene
Locating a gene (or its activity) - Restriction Maps.
Restriction maps... via gel electrophoresis &DNA-electropherogram
DNA fingerprintg. CSI Miami - how to make one: a murder case&a rape case + DNA prints in
Health & Society& DNA Forensic Science
DNA Probe Hybridizationg - to detect specific DNA with a probe
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Comparing Restriction Fragments to a probe:
Southerng Blotting - DNA electrophoresis & blotting one can detect specific gene sequence in
samples by binding to labeled probes
DNA micro-arrays - monitor gene expression in thousands of genes & changes by passing
cDNA of the cell's mRNA over slide with ssDNA of all cell's genes;
DNA microchips are fabricated by high speed robotics akin to Intel chip making
DNA (mRNA's) are fluorescently tagged so easy to see in slide's wells [microchips arrays made
simultaneously by phopshoramidite method of Caruthers].
Gene Sequencing strategy - random fragments are sequenced and then ordered relative to each
other via overlap & supercomputing
Sequencing Strategies methodology dideoxy procedure (development by Fred Sanger)