Download Lecture Presentation to accompany Principles of Life

Genomes & Biotechnology
Key Concepts
• There Are Powerful Methods for
Sequencing Genomes and Analyzing
Gene Products
• Prokaryotic & Eukaryotic Genomes have
several things in common, but some key
differences
• The Human Genome Sequence Has Many
Applications
Key Concepts
• Recombinant DNA Can Be Made in the
Laboratory
• DNA Can Genetically Transform Cells and
Organisms
• Genes and Gene Expression Can Be
Manipulated
• Biotechnology Has Wide Applications
There Are Powerful Methods for Sequencing Genomes and
Analyzing Gene Products
The Human Genome Project was proposed in 1986 to determine the
normal sequence of all human DNA.
The publicly funded effort was aided and complemented by privately
funded groups.
Methods used were first developed to sequence prokaryotes and simple
eukaryotes. (Sanger Method)
Strand to be sequenced
Each flask has a different
terminating nucleotide
Fragment lengths
determined by gel
electrophoresis
Replication products
The fragment sequences are put together using larger, overlapping fragments.
There Are Powerful Methods for Sequencing Genomes and
Analyzing Gene Products
Next-generation DNA sequencing
methods (Next gen) developed in the
1990’s use DNA replication and the
polymerase chain reaction (PCR). These
enabled the project to be finished much
quicker than originally anticipated, 2003.
Figure 12.1 DNA Sequencing (Part 1)
One approach to next-generation DNA sequencing:
•DNA is cut into 100 bp fragments.
•DNA is denatured by heat, and each single strand
then acts a template for synthesis.
•Each fragment is attached to adapter sequences
and then to supports.
•Fragments are then amplified by PCR.
Amplified DNA attached to a solid substrate is
ready for sequencing:
•Fragments are denatured and primers,
DNA polymerase, and fluorescently labeled
nucleotides are added.
•DNA is replicated by adding one nucleotide
at a time.
•Fluorescent color of the particular
nucleotide is detected as it is added,
indicating the sequence of the DNA.
There Are Powerful Methods for Sequencing Genomes and
Analyzing Gene Products
Determining sequences is possible
because original DNA fragments are
overlapping.
Example: A 10 bp fragment cut three
different ways yields
TG, ATG, and CCTAC
AT, GCC, and TACTG
CTG, CTA, and ATGC
The correct sequence is ATGCCTACTG.
Figure 12.2 Arranging DNA Sequences
For genome sequencing the fragments
are called “reads.” Using complex
mathematics and computer programs
further increases the speed at which
“reads” are processed. A field known as
bioinformatics.
There Are Powerful Methods for Sequencing Genomes and
Analyzing Gene Products
The power of this method derives from the
fact that:
• It is fully automated and miniaturized.
• Millions of different fragments are
sequenced at the same time. This is called
massively parallel sequencing.
• It is an inexpensive way to sequence large
genomes.
There Are Powerful Methods for Sequencing Genomes and
Analyzing Gene Products
It is one thing to know the order of the nucleotide
bases, it is another to know what it all means….
In functional genomics, sequences identify the
functions of various parts:
• Open reading frames—the coding regions of the genes, recognized by start and stop
codons for translation, and sequences indicating location of introns
• Amino acid sequences of proteins
• Regulatory sequences—promoters and terminators for transcription
• RNA genes, including rRNA, tRNA, small nuclear RNA, and microRNA genes
• Other noncoding sequences in various categories
Comparative genomics compares a newly
sequenced genome with sequences from other
organisms.
•
It provides information about function of sequences and can trace evolutionary
relationships.
There Are Powerful Methods for Sequencing Genomes and
Analyzing Gene Products
The proteome is the total of the
proteins produced by an
organism—more complex than
its genome.
Many genes encode for more
than one protein, through
alternative splicing and
posttranslational modifications.
Proteomics seeks to identify
and characterize all of the
expressed proteins.
There Are Powerful Methods for Sequencing Genomes and
Analyzing Gene Products
The metabolome is the description of all of
the metabolites of a cell or organism:
• Primary metabolites are involved in normal processes, such
as in pathways like glycolysis. Also includes hormones and
other signaling molecules.
• Secondary metabolites are often unique to particular
organisms or groups.
Examples: Antibiotics made by microbes, and chemicals
made by plants for defense.
Metabolomics aims to describe the metabolome of a tissue
or organism under particular environmental conditions.
Analytical instruments can separate molecules with different
chemical properties, and other techniques can identify them.
Measurements can be related to physiological states.
Figure 12.5 Genomics, Proteomics, and Metabolomics
Prokaryotic & Eukaryotic Genomes have several things in
common, but some key differences
Comparing genomes of prokaryotes and
eukaryotes:
Certain genes are present in all organisms
(universal genes); and some universal
gene segments are present in many
organisms.
This suggests that a minimal set of DNA
sequences is common to all cells.
Prokaryotic & Eukaryotic Genomes have several things in
common, but some key differences
Efforts to define a minimal genome of life
involve computer analysis of genomes, the
study of the smallest known genome (M.
genitalium), and using transposons as
mutagens.
Transposons can insert into genes at
random; the mutated bacteria are tested
for growth and survival, and DNA is
sequenced.
Prokaryotic & Eukaryotic Genomes have several things in common, but some
key differences
Transposons are of two main types in eukaryotes:
Retrotransposons (Class I) make RNA copies of
themselves, which are copied into DNA and
inserted in the genome.
 LTR retrotransposons have long terminal repeats of
DNA sequences
 Non-LTR retrotransposons do not have LTR sequences
DNA transposons (Class II) do not use RNA
intermediates.
They are excised from the original location and inserted at a
new location without being replicated.
Prokaryotic & Eukaryotic Genomes have several things in
common, but some key differences
Transposons (or transposable
elements) are DNA segments
that can move from place to
place in the genome.
They can move from one piece of
DNA (such as a chromosome),
to another (such as a plasmid).
If a transposon is inserted into the
middle of a gene, it will be
transcribed and result in
abnormal proteins.
If a small transposon is duplicated
and the 2 copies are then
separated by host genes, the
whole complex can be carried
to other locations within the
genome. This can result in
multiple copies of a gene.
Prokaryotic & Eukaryotic Genomes have several things in common, but some
key differences
A group of closely related genes are called gene
families .
These arose over evolutionary time when different copies of
genes underwent separate mutations.
For example: Genes encoding the globin proteins in
hemoglobin and myoglobin all arose from a single common
ancestral gene.
Many gene families include nonfunctional
pseudogenes (Ψ), resulting from mutations that
cause a loss of function, rather a new one.
A pseudogene may simply lack a promoter, and thus fail to be
transcribed, or a recognition site, needed for the removal of
an intron.
Prokaryotic & Eukaryotic Genomes have several things in common, but some
key differences
Eukaryotic genomes have repetitive DNA sequences:
• Highly repetitive sequences—short sequences (< 100 bp)
repeated thousands of times in tandem; not transcribed
• Short tandem repeats (STRs) of 1–5 bp are scattered around the
genome and can be used in DNA fingerprinting.
• Moderately repetitive sequences are repeated 10–1,000
times.
 Includes the genes for tRNAs and rRNAs
 Single copies of the tRNA and rRNA genes are
inadequate to supply large amounts of these molecules
needed by cells, so genome has multiple copies in
clusters
Most moderately repeated sequences are transposons.
Table 12.3 Types of Sequences in Eukaryotic Genomes
Prokaryotic & Eukaryotic Genomes have several things in
common, but some key differences
Features of bacterial and
archaeal genomes:
Features of eukaryote genomes:
Relatively small, with single,
circular chromosome
Much larger, linear, several
chromosomes
Compact—mostly protein-coding
regions
Mostly non-protein – coding
regions but have more protein
coding regions overall
Most do not contain introns
Contain introns, gene control
sequences, and repeated
sequences
Often carry plasmids, smaller
circular DNA molecules
Do not contain plasmids but
contain more regulatory genes
Prokaryotic & Eukaryotic Genomes have several things in
common, but some key differences
Several model
organisms have been
studied and used
extensively.
Model organisms are
easy to grow and
study in a laboratory,
their genetics are well
studied, and their
characteristics
represent a larger
group of organisms.
Prokaryotic & Eukaryotic Genomes have several things in
common, but some key differences
Prokaryotes can be identified
by their growth in culture,
but DNA can also be
isolated directly from
environmental samples.
DNA can then be cloned for
“libraries” or amplified and
sequenced to detect known
and unknown organisms.
E. coli is often used to “store”
the library of genes.
The Human Genome Sequence Has Many Applications
By 2010 the complete haploid genome sequence
was completed for more than ten individuals.
Now scientists are working on the 1000 project:
The 1000 Genomes Project is an international collaboration to produce an
extensive public catalog of human genetic variation, including SNPs and
structural variants, and their haplotype contexts. This resource will support
genome-wide association studies and other medical research studies.
The genomes of about 2500 unidentified people from about 25 populations
around the world will be sequenced using next-generation sequencing
technologies. The results of the study will be freely and publicly accessible
to researchers worldwide.
http://www.1000genomes.org/
The average person can also explore facets of their
own DNA for as little as a $100.
The Human Genome Sequence Has Many Applications
Some interesting facts about the human genome:
• Protein-coding genes make up about 24,000 genes, less than 2
percent of the 3.2 billion base pair human genome.
• Each gene must code for several proteins, and posttranscriptional
mechanisms (e.g., alternative splicing) must account for the
observed number of proteins in humans.
• An average gene has 27,000 base pairs, but size varies greatly as
does the size of the proteins.
• All human genes have many introns.
• 3.5 percent of the genome is functional but noncoding—have roles
in gene regulation (microRNAs) or chromosome structure.
• Over 50 percent of the genome is transposons and other repetitive
sequences.
• Most of the genome (97 percent) is the same in all people.
• Chimpanzees share 95 percent of the human genome.
Figure 12.9 Functions of the Eukaryotic Genome
Figure 12.12 Evolution of the Genome
The Human Genome Sequence Has Many Applications
Rapid genotyping technologies are being
used to understand the complex genetic
basis of diseases such as diabetes, heart
disease, and Alzheimer’s disease.
“Haplotype maps” are based on single
nucleotide polymorphisms (SNPs)—
DNA sequence variations that involve
single nucleotides.
SNPs are point mutations in a DNA
sequence.
The Human Genome Sequence Has Many Applications
SNPs that differ are not all inherited as
independent alleles.
A set of SNPs that are close together on a
chromosome are inherited as a linked unit.
A piece of chromosome with a set of linked
SNPs is called a haplotype.
Analyses of human haplotypes have shown
that there are, at most, 500,000 common
variations.
The Human Genome Sequence Has Many Applications
Technologies to analyze SNPs in an
individual genome include next-generation
sequencing methods and DNA
microarrays.
A DNA microarray detects DNA or RNA
sequences that are complementary to and
hybridize with an oligonucleotide probe.
The aim is to find out which SNPs are
associated with specific diseases and
identify alleles that contribute to disease.
Figure 12.13 SNP Genotyping and Disease
The Human Genome Sequence Has Many Applications
Genetic variation can
affect an individual’s
response to a
particular drug.
A variation could make
an drug more or less
active in an
individual.
Pharmacogenomics
studies how the
genome affects the
response to drugs.
This makes it possible
to predict whether a
drug will be effective,
with the objective of
personalizing drug
treatments.
The Human Genome Sequence Has Many Applications
DNA fingerprinting refers
to a group of techniques
used to identify
individuals by their DNA.
Short tandem repeat
(STR) analysis is most
common.
When several different
STR loci are analyzed, a
unique pattern becomes
apparent.
Can be used for questions
of paternity and in crime
investigation
Recombinant DNA Can Be Made in the Laboratory
It is possible to modify organisms with genes from other,
distantly related organisms.
Recombinant DNA is a DNA molecule made in the laboratory
that is derived from at least two genetic sources.
Three key tools:
• Restriction enzymes for cutting DNA into fragments
• Gel electrophoresis for analysis and purification of DNA
fragments
• DNA ligase for joining DNA fragments together in new
combinations
Recombinant DNA Can Be Made in the Laboratory
Restriction enzymes recognize a specific DNA sequence
called a recognition sequence or restriction site.
5′…….GAATTC……3′
3′…….CTTAAG……5′
Each sequence forms a palindrome: the opposite strands
have the same sequence when read from the 5′ end.
Some restriction enzymes create a blunt cut in DNA leaving a
short sequence of single-stranded DNA at each end.
Staggered cuts result in overhangs, or “sticky ends;” straight
cuts result in “blunt ends.”
Sticky ends can bind complementary sequences on other
DNA molecules.
Recombinant DNA Can Be Made in the Laboratory
DNA fragments cut by enzymes can be
separated by gel electrophoresis.
Negatively charged DNA fragments move towards the positive
end.
Smaller fragments move faster than larger ones.
DNA fragments separate and give three types of information:
• The number of fragments
• The sizes of the fragments
• The relative abundance of the fragments, indicated by the
intensity of the band
Recombinant DNA Can Be Made in the Laboratory
After separation on a gel,
a specific DNA
sequence can be found
with a complementary
single-stranded probe.
The gel region can be cut
out and the DNA
fragment removed.
The purified DNA can be
analyzed by sequence
or used to make
recombinant DNA.
With restriction enzymes
to cut fragments and
DNA ligase to combine
them, new recombinant
DNA can be made.
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
Recombinant DNA technology can be used to clone (make
identical copies) genes.
Transformation: Recombinant DNA is cloned by inserting it
into host cells (transfection if host cells are from an
animal).
The altered host cell is called transgenic.
Usually only a few cells exposed to recombinant DNA are
actually transformed.
To determine which of the host cells are transgenic, the
recombinant DNA includes selectable marker genes, such
as genes that confer resistance to antibiotics. (Refer to
bacterial transformation lab)
Concept 13.2 DNA Can Genetically Transform Cells and
Organisms
Selectable markers are a type of reporter gene—a gene
whose expression is easily observed.
Green fluorescent protein, which normally occurs in a jellyfish,
emits visible light when exposed to UV light.
The gene for this protein has been isolated and incorporated
into vectors as a reporter gene.
DNA Can Genetically Transform Cells and Organisms
Methods for inserting the recombinant DNA into a cell:
• Cells may be treated with chemicals to make plasma
membranes more permeable—DNA diffuses in. (CaCl2)
• Electroporation—a short electric shock creates temporary
pores in membranes, and DNA can enter.
• Biological Vector - Viruses and bacteria can be altered to
carry recombinant DNA into cells.
• Mechanical Vector - “Gene guns” can “shoot” the host cells
with particles of DNA.
• Transgenic animals can be produced by injecting
recombinant DNA into the nuclei of fertilized eggs.