Download DNA Sequencing

Chapter 12. DNA Technology
The DNA Toolbox
• Sequencing of the human genome was completed by
2007.
• DNA sequencing has depended on advances in
technology, starting with making recombinant DNA.
• In recombinant DNA, nucleotide sequences from two
different sources, often two species, are combined in
vitro into the same DNA molecule.
• Methods for making recombinant DNA are central to
genetic engineering, the direct manipulation of genes
for practical purposes.
• DNA technology has revolutionized biotechnology, the
manipulation of organisms or their genetic components
to make useful products.
DNA cloning yields multiple copies of
a gene or other DNA segment
• To work directly with specific genes, scientists prepare
gene-sized pieces of DNA in identical copies, a process
called DNA cloning.
DNA Cloning and Its Applications
• Most methods for cloning pieces of DNA in the
laboratory share general features, such as the use of
bacteria and their plasmids.
• Plasmids are small circular DNA molecules that replicate
separately from the bacterial chromosome.
• Cloned genes are useful for making copies of a
particular gene and producing a protein product.
• Gene cloning involves using bacteria to make multiple
copies of a gene.
• Foreign DNA is inserted into a plasmid, and the
recombinant plasmid is inserted into a bacterial cell.
• Reproduction in the bacterial cell results in cloning of
the plasmid including the foreign DNA.
• This results in the production of multiple copies of a
single gene.
DNA Cloning
Bacterium
1 Gene inserted into
Cell containing gene
of interest
plasmid
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Gene of
interest
2
2 Plasmid put into
bacterial cell
Recombinant
bacterium
DNA of
chromosome
Recombinant
bacterium
3 Host cell grown in culture
to form a clone of cells
containing the “cloned”
gene of interest
Protein expressed
by gene of interest
Gene of
Interest
Copies of gene
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
4 Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
Using Restriction Enzymes to Make
Recombinant DNA
• Bacterial restriction enzymes (제한효소) cut DNA
molecules at specific DNA sequences called restriction
sites.
• A restriction enzyme usually makes many cuts, yielding
restriction fragments.
• The most useful restriction enzymes cut DNA in a
staggered way, producing fragments with “sticky ends”
that bond with complementary sticky ends of other
fragments.
• DNA ligase is an enzyme that seals the bonds between
restriction fragments.
Restriction site
DNA 5
3
1
3
5
Restriction enzyme
(BamHI) cuts sugarphosphate backbones.
Sticky end
2
DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
One possible combination
3
DNA ligase
seals strands.
Recombinant DNA molecule
Cloning a Eukaryotic Gene in a
Bacterial Plasmid
• In gene cloning, the original plasmid is called a cloning
vector.
• A cloning vector is a DNA molecule that can carry
foreign DNA into a host cell and replicate there.
Hummingbird
cell
TECHNIQUE
Bacterial cell
lacZ gene
ampR gene Bacterial
Restriction
site
Sticky
ends
plasmid
Gene of interest
Hummingbird
DNA fragments
Nonrecombinant
plasmid
Recombinant plasmids
Bacteria carrying
plasmids
RESULTS
Colony carrying nonrecombinant plasmid
with intact lacZ gene
Colony carrying recombinant
plasmid with disrupted lacZ gene
One of many
bacterial
clones
Storing Cloned Genes in DNA
Libraries
• A genomic library that is made using bacteria is the
collection of recombinant vector clones produced by
cloning DNA fragments from an entire genome.
• A genomic library that is made using bacteriophages is
stored as a collection of phage clones.
Foreign genome
cut up with
restriction
enzyme
or
Recombinant
phage DNA
Bacterial
clones
(a) Plasmid library
Recombinant
plasmids
Phage
clones
(b) Phage library
• A bacterial artificial chromosome (BAC) is a large
plasmid that has been trimmed down and can carry a
large DNA insert.
• BACs are another type of vector used in DNA library
construction.
Large plasmid
Large insert
with many genes
BAC
clone
(c) A library of bacterial artificial
chromosome (BAC) clones
• A complementary DNA (cDNA) library is made by
cloning DNA made in vitro by reverse transcription of all
the mRNA produced by a particular cell.
• A cDNA library represents only part of the genome —
only the subset of genes transcribed into mRNA in the
original cells.
DNA in
nucleus
mRNAs in
cytoplasm
mRNA
Reverse
transcriptase
Poly-A tail
DNA Primer
strand
Degraded
mRNA
DNA
polymerase
cDNA
Screening a Library for Clones
Carrying a Gene of Interest
• A clone carrying the gene of interest can be identified
with a nucleic acid probe having a sequence
complementary to the gene.
• This process is called nucleic acid hybridization.
• A probe can be synthesized that is complementary to
the gene of interest.
• For example, if the desired gene is
5
… G G C T A A C T T AG C …
– Then we would synthesize this probe
3 C C G A T T G A A T C G 5
3
Radioactive
DNA probe
Single-stranded
DNA
Mix with singlestranded DNA from
genomic library
Base pairing
indicates the
gene of interest
· The DNA probe can be used to screen a large number of clones
simultaneously for the gene of interest.
· Once identified, the clone carrying the gene of interest can be
cultured.
TECHNIQUE
Radioactively
labeled probe
molecules
Multiwell plates
holding library
clones
Probe
DNA
Gene of
interest
Single-stranded
DNA from cell
Film
•
Nylon membrane
Nylon
Location of
membrane
DNA with the
complementary
sequence
Amplifying DNA in Vitro: The
Polymerase Chain Reaction (PCR)
• The polymerase chain reaction, PCR, can produce many
copies of a specific target segment of DNA.
• A three-step cycle — heating, cooling, and replication —
brings about a chain reaction that produces an
exponentially growing population of identical DNA
molecules.
TECHNIQUE
Genomic DNA
1
2
Cycle 1
yields
2
molecules
5
3
3
Target
sequence
5
Denaturation 5
3
3
5
Annealing
Primers
3
Extension
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
PCR Process
5
TECHNIQUE
3
Target
sequence
Genomic DNA
3
5
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
New
nucleotides
Cycle 1
yields 2 molecules
Genomic
DNA
3
5
5
3
3
5
1 Heat to
separate
DNA strands
Target
sequence
3
5
5
2 Cool to allow
primers to form
hydrogen bonds
with ends of
target sequences
3
5
3 DNA
polymerase adds
nucleotides
to the 3 end
of each primer
5
5
3
5
3
Primer
5
New DNA
3
Cycle 2
yields 4 molecules
Cycle 3
yields 8 molecules
DNA technology allows us to study
the sequence, expression, and
function of a gene
• DNA cloning allows researchers to
– Compare genes and alleles between individuals.
– Locate gene expression in a body.
– Determine the role of a gene in an organism.
• Several techniques are used to analyze the DNA of
genes.
Gel Electrophoresis and Southern
Blotting
• One indirect method of rapidly analyzing and comparing
genomes is gel electrophoresis.
• This technique uses a gel as a molecular sieve to
separate nucleic acids or proteins by size.
• A current is applied that causes charged molecules to
move through the gel.
• Molecules are sorted into “bands” by their size.
TECHNIQUE
Mixture of DNA molecules
of different sizes
Power
source
Anode +
– Cathode
Gel
1
Power
source
–
+
Longer
molecules
2
Shorter
molecules
Mixture of DNA
fragments of
different sizes
Power
source
Longer
(slower)
molecules
Gel
Completed gel
Shorter
(faster)
molecules
• A technique called Southern blotting combines gel
electrophoresis of DNA fragments with nucleic acid
hybridization.
• Specific DNA fragments can be identified by Southern
blotting, using labeled probes that hybridize to the DNA
immobilized on a “blot” of gel .
TECHNIQUE
DNA + restriction enzyme
Restriction
fragments
I
II
III
Nitrocellulose
membrane (blot)
Heavy
weight
Gel
Sponge
I Normal
II Sickle-cell
-globin allele
allele
III Heterozygote
1 Preparation of restriction fragments
Alkaline
solution
2 Gel electrophoresis
Paper
towels
3 DNA transfer (blotting)
Radioactively labeled
probe for -globin gene
I
II
III
Probe base-pairs
with fragments
Fragment from
sickle-cell
-globin allele
Fragment from
normal -globin
Nitrocellulose blot
allele
4 Hybridization with radioactive probe
I
II
III
Film
over
blot
5 Probe detection
RFLPs can be used to detect
differences in DNA sequences
– Single nucleotide polymorphism (SNP) is a
variation at one base pair within a coding or
noncoding sequence
– Restriction fragment length polymorphism
(RFLP) is a variation in the size of DNA
fragments due to a SNP that alters a restriction
site
• In restriction fragment analysis, DNA fragments
produced by restriction enzyme digestion of a DNA
molecule are sorted by gel electrophoresis.
• Restriction fragment analysis is useful for comparing
two different DNA molecules, such as two alleles for a
gene.
• The procedure is also used to prepare pure samples of
individual fragments.
Normal -globin allele
175 bp
DdeI
201 bp
DdeI
Normal Sickle-cell
allele
allele
Large fragment
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
Large fragment
376 bp
DdeI
DdeI
DdeI
(a) DdeI restriction sites in normal and
sickle-cell alleles of -globin gene
201 bp
175 bp
376 bp
(b) Electrophoresis of restriction fragments
from normal and sickle-cell alleles
Restriction
enzymes added
DNA sample 1
DNA sample 2
w
Cut
z
x
Cut
Cut
y
y
Longer
fragments
z
x
Shorter
fragments
w
y
y
Forensic Evidence and Genetic
Profiles
• An individual’s unique DNA sequence, or genetic profile,
can be obtained by analysis of tissue or body fluids.
• Genetic profiles can be used to provide evidence in
criminal and paternity cases and to identify human
remains.
• Genetic profiles can be analyzed using RFLP analysis by
Southern blotting.
• Even more sensitive is the use of genetic markers called short
tandem repeats (STRs), which are variations in the number of
repeats of specific DNA sequences.
• STR analysis compares the lengths of STR sequences at
specific regions of the genome
• PCR and gel electrophoresis are used to amplify and then
identify STRs of different lengths.
• The probability that two people who are not identical twins
have the same STR markers is exceptionally small.
• Current standard for DNA profiling is to analyze 13 different
STR sites.
STR site 1
STR site 2
Crime scene DNA
Number of short tandem
repeats match
Suspect’s DNA
Number of short tandem
repeats do not match
Crime scene
DNA
Suspect’s
DNA
(a) This photo shows Earl Washington
just before his release in 2001,
after 17 years in prison.
Source of
sample
STR
marker 1
STR
marker 2
STR
marker 3
Semen on victim
17, 19
13, 16
12, 12
Earl Washington
16, 18
14, 15
11, 12
Kenneth Tinsley
17, 19
13, 16
12, 12
(b) These and other STR data exonerated Washington and
led Tinsley to plead guilty to the murder.
DNA profiling has provided evidence in
many forensic investigations
●
Forensics
– Evidence to show guilt or innocence
●
Establishing family relationships
– Paternity analysis
●
Identification of human remains
– After tragedies such as the September 11, 2001, attack
on the World Trade Center
●
Species identification
– Evidence for sale of products from endangered species
DNA Sequencing
• Relatively short DNA fragments can be sequenced by the
dideoxy chain termination method.
• Modified nucleotides called dideoxyribonucleotides
(ddNTP) attach to synthesized DNA strands of different
lengths.
• Each type of ddNTP is tagged with a distinct fluorescent
label that identifies the nucleotide at the end of each
DNA fragment.
• The DNA sequence can be read from the resulting
spectrogram.
TECHNIQUE
DNA
(template strand)
Primer
DNA
polymerase
Deoxyribonucleotides
Dideoxyribonucleotides
(fluorescently tagged)
dATP
ddATP
dCTP
ddCTP
dTTP
ddTTP
dGTP
ddGTP
TECHNIQUE
DNA (template
strand)
Labeled strands
Shortest
Direction
of movement
of strands
Longest
Longest labeled strand
Detector
Laser
RESULTS
Shortest labeled strand
Last base
of longest
labeled
strand
Last base
of shortest
labeled
strand
그림 12.15
그림 12.15 DNA 염기서열 분석
Genomics is the scientific study of whole
genomes
●
Genomics is the study of an organism’s
complete set of genes and their interactions
– Initial studies focused on prokaryotic genomes
– Many eukaryotic genomes have since been
investigated
●
Evolutionary relationships can be elucidated
– Genomic studies showed a 96% similarity in DNA
sequences between chimpanzees and humans
– Functions of human disease-causing genes have
been determined by comparisons to similar genes
in yeast
The Human Genome Project revealed that
most of the human genome does not
consist of genes
– Goals of the Human Genome Project (HGP)
– To determine the nucleotide sequence all DNA in
the human genome
– To identify the location and sequence of every
human gene
Results of the Human Genome Project
Humans have ~21,000 genes in 3.2 billion nucleotide
pairs
● Only ~1.5% of the DNA codes for proteins, tRNAs, or
rRNAs
● The remaining 88.5% of the DNA contains
– Control regions such as promoters and enhancers
– Unique noncoding DNA
– Repetitive DNA
– Found in centromeres and telomeres
– Found dispersed throughout the genome,
related to transposable elements that can
move or be copied from one location to
another
●
Exons (regions of genes coding for protein
or giving rise to rRNA or tRNA) (1.5%)
Repetitive
DNA that
includes
transposable
elements
and related
sequences
(44%)
Introns and
regulatory
sequences
(24%)
Unique
noncoding
DNA (15%)
Repetitive
DNA
unrelated to
transposable
elements
(15%)
The whole-genome shotgun method of
sequencing a genome can provide a
wealth of data quickly
Three stages of the Human Genome Project
●
– A low-resolution linkage map was developed using
RFLP analysis of 5,000 genetic markers
– A physical map was constructed from nucleotide
distances between the linkage-map markers
– DNA sequences for the mapped fragments were
determined
Whole-genome Shotgun Method
●
●
Restriction enzymes were used to produce fragments
that were cloned and sequenced
Computer analysis assembled the sequence by
aligning overlapping regions
Chromosome
Chop up with
restriction enzyme
DNA fragments
Sequence
fragments
Align
fragments
Reassemble
full sequence
Proteomics is the scientific study of the
full set of proteins encoded by a
genome
●
Proteomics
– Studies the proteome, the complete set of proteins
specified by a genome
– Investigates protein functions and interactions
●
The human proteome may contain 100,000
proteins
Analyzing Gene Expression
• Nucleic acid probes can hybridize with mRNAs
transcribed from a gene.
• Probes can be used to identify where or when a gene is
transcribed in an organism.
Studying the Expression of Single
Genes
• Changes in the expression of a gene during embryonic
development can be tested using
– Northern blotting.
– Reverse transcriptase-polymerase chain reaction.
– In situ hybridization.
• The methods are used to compare mRNA from different
developmental stages.
• Northern blotting combines gel electrophoresis of
mRNA followed by hybridization with a probe on a
membrane.
• Identification of mRNA at a particular developmental
stage suggests protein function at that stage
• Reverse transcriptase-polymerase chain reaction (RTPCR) is quicker and more sensitive.
• Reverse transcriptase is added to mRNA to make cDNA,
which serves as a template for PCR amplification of the
gene of interest.
• The products are run on a gel and the mRNA of interest
identified
TECHNIQUE
1 cDNA synthesis
mRNAs
cDNAs
2 PCR amplification
Primers
-globin
gene
3 Gel electrophoresis
RESULTS
Embryonic stages
1
2
3
4
5
6
• In situ hybridization uses fluorescent dyes attached to
probes to identify the location of specific mRNAs in
place in the intact organism.
Studying the Expression of
Interacting Groups of Genes
• Automation has allowed scientists to measure expression
of thousands of genes at one time using DNA
microarray assays.
• DNA microarray assays compare patterns of gene
expression in different tissues, at different times, or
under different conditions.
TECHNIQUE
1 Isolate mRNA.
Tissue sample
2 Make cDNA by reverse
mRNA molecules
3 Apply the cDNA mixture to a
Labeled cDNA molecules
(single strands)
DNA fragments
representing
specific genes
transcription, using
fluorescently labeled
nucleotides.
microarray, a different gene in
each spot. The cDNA hybridizes
with any complementary DNA on
the microarray.
DNA microarray
4 Rinse off excess cDNA; scan
microarray for fluorescence.
Each fluorescent spot represents a
gene expressed in the tissue sample.
DNA microarray
with 2,400
human genes
Green Fluorescent Protein
• The green fluorescent protein (GFP) of the jellyfish
Aequorea victoria has become one of the most
important fluorescent labels in biology.
Its Applications as a Biomarker
Monitoring Cellular Proteins