Download Chapter 20 DNA Technology and Genomics

Chapter 20 DNA Technology and
Genomics
Manipulating Genomes
• Genetic engineering- direct manipulation of
genes for practical purposes
Ex: hormones, blood clotting factors,
wine and cheese making, selective
breeding of livestock,
- recombinant DNA – DNA with nucleotide
sequences from 2 different sources
combined in vitro
DNA Sequencing
• Ultimate goal of mapping is
determining nucleotide
sequences for each
chromosome
• Steps of approach:
1. Cut DNA from many
copies of chromosome
into overlapping
fragments
2. Clone fragments in
plasmid or phage vectors
3. Sequence fragments
4. Order sequence into
overall sequence
Gene Cloning
• Gene cloning – methods for preparing gene-sized
pieces of DNA in multiple copies
- has allowed scientists to work directly with
specific genes
- 2 basic purposes
1. make many copies of a specific gene
2. produce a protein product
Ex: pest resistance in plants &
medical uses in large quantity
(GH)
Overview of Gene Cloning
• Plasmid – small ring
of DNA that carries
accessory genes
separate from
bacterial
chromosome
• Cloning vectors –
original plasmid that
can carry foreign
DNA into a cell and
replicate there
Producing Clones of Cells
Steps to Cloning with a bacterial plasmid:
1. Isolate the bacterial plasmid and DNA with the gene of
interest.
2. Both the plasmid and the DNA are digested by the same
restriction enzyme. The plasmid is cut at one specific site,
while the DNA is cut many times creating fragments.
3. Allow mixing of DNA fragments with plasmids and
matching of complementary sticky ends. DNA ligase is
added to seal the strands together.
4. The new DNA is mixed with mutated bacteria
5. Cloning begins by plating out bacteria on nutrient agar
with the target substance.
- colonies will be identified by ability to reproduce in
the agar & by distinct color
Restriction Endonucleases
• Restriction enzymes- cut
DNA molecules at a limited
number of specific locations
-normally protect cell
from intruding DNA
from other organisms
-enzymes are very
specific (restriction site)
- sets of restriction
fragments are
determined by specific
enzyme
Gene Identification
• Nucleic acid hybridization – DNA of a gene
can be detected by its ability to base-pair with
a complementary sequence of another nucleic
acid molecule
• Genomic Library – complete set of plasmid
clones each carrying copies of a particular
segment from initial genome
Gel Electrophoresis
• Technique using a gel as a molecular sieve to
separate nucleic acids or proteins on basis of
size, electrical charge, and physical properties.
-nucleic acids have negative charge from
phosphate group and will travel to
positive end
-sorts DNA fragments produced by
restriction enzyme digestion
Polymerase Chain Reaction (PCR)
• PCR – quicker more selective method in which
any target segment of DNA can be quickly
amplified.
*billions of copies can be made in a few
hours
*high speed and high accuracy
*small amounts needed to start process
*primers are very specific (Taq
polymerase)
PCR
• Sources possible for
DNA amplification:
a. 40,000 year old
woolly mammoth
b. Tiny amounts of
blood, tissue, and
semen from crime
scenes
c. single embryonic
cells
d. DNA in viral
genes
Eukaryotic Cloning
• Yeast is an ideal cloning eukaryotic genes
- easy to grow as bacteria
- contains plasmids ( rare in eukaryotes)
- solves incompatibility with prokaryotes
• Yeast Artificial Chromosomes (YACs) – combine
essentials of eukaryotic chromosome such as
origin of DNA replication, centromere, and 2
telomeres with foreign DNA
- advantage comes from the longer DNA
segment carried that is more likely to contain
entire gene rather than a portion
Southern Blotting
• Technique combines gel electrophoresis and
nucleic acid hybridization
*reveals whether a particular sequence is
present in a sample and also the size of
restriction fragments that contain the
sequence
* can also identify heterozygote carriers of
mutant alleles associated with genetic
disorders
Restriction Fragment Analysis
• Restriction Fragment analysis – detects certain
differences in nucleotide sequences of DNA molecules
Questions answered by this technique:
1. Does a particular gene vary from person to
person?
2. Are certain alleles associated with hereditary
disorders?
3. Where in the body and when is it expressed?
4. Where is gene located within genome?
RFLPs
• Restriction fragment length polymorphisms –
caused by differences in restriction fragment
patterns from different restriction sites on
homologous chromosomes
-serves as a genetic marker for a particular
location in the genome
-increased the number of markers available for
mapping the human genome
Genomics
• Study of whole sets of genomes and their
interactions
*more direct study of genes without inferring
genotype from phenotype
*Genome size generally increases from
prokaryotes to eukaryotes, but does not correlate
in biological complexity
Ex: 25,000 genes in human vs. 50,000 to
100,000 found in fruit fly or worm
Gene Functions
• In vitro mutagenesis- introduces a mutation in
to the sequence and disables the gene or may
destroy the function of the product.
-Observations may reveal true function of
normal gene
• RNA interference – utilizes double stranded
RNA molecules that will match a particular
gene sequence and trigger the breakdown or
block translation
Gene Functions
• Proteomics- study of full protein sets encoded by
genomes
-number of proteins carrying out activities of
the cell outnumbers the number of genes
-Knowing when and where is vital
• Single nucleotide polymorphisms- single base
pair variations in genome
occur about 1 in 1000 base pairs
-several million sites might be possible which
accounts for a .1% difference between DNA
sequences of like humans
20.3 Cloning and Stem Cells
Organismal cloning – cloning whole multi-cellular
organisms from a single cell
- genetically identical to parent
-generates stem cells
Stem cell – unspecialized cell that can reproduce
itself and differentiate into one or more specialized
cells
- potential for regenerating damaged tissues
Cloning Plants
• First successfully accomplished in the 1950’s with
carrot plants
• Results showed that differentiation does not
involve irreversible changes in DNA
totipotent – ability of mature cells to
“dedifferentiate” and give rise to all
specialized cell types
Ex: orchids cloned for commercial
purposes
- pathogen resistance also possible
Cloning Animals
• Differentiated cells in animals do not divide in culture –
different approach to tell if animal cells were totipotent
• Nuclear transplantation – removal of nucleus of an
unfertilized or fertilized egg and replace it with a
nucleus of a differentiated cell
- In studies with tadpoles it was found that the
potential of transplanted nucleus to direct
normal development was inversely related to the
age of the donor
- older the donor the lower % of normally
developing tadpoles
Ex: Dolly the sheep – identical to donor age
6, suffered from arthritis and lung disease
seen in older sheep
Faulty Gene Regulation
• In most nuclear transplantation studies only a
small % of cloned embryos develop normally
Reasons for low efficiency and high defects:
1. Gene regulation as a result of epigenetic
changes in chromatin
2. Misplaced or extra methyl groups that
contribute methylation pattern of expressed or
repressed genes
3. Reprogramming of donor nuclei and
restructuring of chromatin to match newly
fertilized egg
Stem Cells
• Main reasons for cloning embryos is not for
reproduction, but for production of stem cells to treat
human diseases
• Embryonic stem cells (ES) – stem cells isolated from
blastula/blastocyst stages are considered
• Pluripotent – capable of differentiating into many
diverse specialized cell types even eggs or sperm
- ultimate goal is repair for damaged or diseased
organs
Ex: insulin producing pancreatic cells in
Type 1 diabetes patients; or brain cells for
patients with Huntington’s or Parkinson’s
disease
Stem Cells
• Adult stem cells – not as diverse in generating
specialized cells as embryonic but multiple types
Ex: bone marrow stem cells can generate
different blood cells, bone, cartlilage, muscle, and
even nerve cells
- only tiny numbers of adult stem cells
- development is limited to certain tissues
- unlike embryonic cells less political/ethical
issues obtaining them
20.4 Gene Therapy
• Results have been inconclusive in some cases and
led to secondary issues in other cases
• Ethical questions have risen about tampering
with human genes
*Eugenics – deliberate effort to control
genetic makeup of humans.
• Others see no difference between transplanting
somatic cells and transplanting organs
• Other questions center around interfering with
evolution or genetic variation.
Medical Applications
• DNA technology has increased the identification
of genes whose mutants lead to genetic diseases.
-Detection may lead to ways of diagnosing,
treating, and possible preventing these
diseases.
Ex: HIV RNA being detected by RT PCR,
Sickle cell, cystic fibrosis, Huntington’s
disease, hemophilia, and muscular
dystrophy
Gene Therapy
• Gene therapy is the alteration of an afflicted
individual’s genes
-targets disorders that have been traceable
to a single defective gene
-its believed a normal allele could be
inserted into a somatic cell of a defective
gene
-somatic cell must be a cell that divides
throughout patient’s life ( ex: bone marrow)
Pharmaceuticals
• Examples of DNA engineered products:
1. Human insulin – 2 million people in US
with diabetes might be dependent on this
drug
2. Human growth hormone – targets children
with dwarfism from inadequate amounts of
natural HGH
3. Tissue Plasminogen Activator (TPA)- if given
shortly after heart attack will dissolve blood
clots and maybe reduce risk of subsequent
heart attacks
Forensic Evidence
• DNA testing in murder cases can clearly
identify the suspect better than blood or
tissue samples
– RFLP analysis by Southern blotting can serve to
identify similarities and differences in DNA from
small samples
– Provides DNA fingerprint- specific band of
patterns unique to every individual
Agricultural Applications
• Transgenic animals- have gene inserted from another
animal into their genome
-usually in vitro injection after fertilization,
then embryo injected into surrogate mother
- Examples of goals:
1. Create sheep with better quality wool
2. Pigs with leaner meat
3. Shorter time to maturity in cattle
4. Larger muscled cattle for more meat
production
Agricultural Applications
• Ti plasmid- vector method
used to introduce new genes
into crop plants
*Purpose could be to
incorporate pest
resistance, herbicide
resistance, delayed
ripening, or even
increased nutritional
value
Safety and Ethical Questions
• Concerns about DNA technology releasing or
creating new hazardous pathogens led to a set
of guidelines that were adopted as formal
governmental regulations
• Concerns today about hazards center around
Genetically Modified organisms (GMOs).
Ex: salmon cloned and harvested for
growth hormone
Transgenic crops
Safety and Ethical Questions
• Who should have the right to examine
someone else’s genes?
• How should the information be used?
• Should a person’s genome be a factor in
suitability for a job or eligibility for insurance?
• When has it been taken to far?
Mapped Genomes
• Human Genome Project – 1990-2003 completed
when the nucleotide sequence of majority of
DNA in each human chromosome was obtained.
-researchers have also mapped sequences for
E coli, yeast, nematodes, fruit flies and the
mouse
-Progressed in more detail in 3 stages:
1. Genetic mapping
2. Physical mapping
3. DNA sequencing
Genetic Mapping
• linkage maps – several
thousand genetic
markers spaced
throughout each
chromosome
-order and distances
between markers is
based on
recombination
frequencies
-can be genes, RFLPs,
or simple sequence
DNA
Physical Mapping
• Maps the distances between
markers with some physical
measure, usually number of
base pairs along the DNA
- cut DNA of each
chromosome into
restrictions fragments
-determine original
order of fragments
-find overlaps between
fragment ends using
probes