chapter 20 introduction to molecular genetics denniston topping caret 5 th edition copyright the...
TRANSCRIPT
Chapter 20
Introduction to Molecular Genetics
Denniston Topping Caret
5th Edition
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
20.1 The Structure of the Nucleotide
• DNA and RNA are long polymers whose monomer units are called nucleotides
• A nucleotide consists of:1. Nitrogen containing heterocyclic base
• Purine
• Pyrimidine
2. Five-carbon sugar ring • Ribose
• Deoxyribose
3. Phosphoryl group
Nucleotide Structure• Ring structures are found in
both the base and the sugar– Base rings are numbered as
usual– Sugar ring numbers are given
the designation ' or prime• Covalent bond between the
sugar and the phosphoryl group is a phosphoester bond
• Bond between the base and the sugar is a -N-glycosidic linkage joining the 1'-carbon of the sugar and a nitrogen atom of the base
20.1
The
Str
uctu
re o
f th
e N
ucle
otid
e
Major Purine Bases
NCCH
C
N
NC
CH
N
NH2
HNC
CHC
N
NC
C
NH
O
HNH2
12
3 4
56 7
89
Adenine Guanine20.1
The
Str
uctu
re o
f th
e N
ucle
otid
eNitrogenous bases are heterocyclic amines
– Cyclic compounds with at least 1 N atom in the ring structure
– Purines are a double ring structure• A 6-member ring fused to a 5-member ring
Major Pyrimidine Bases
NC CHNH
O
CCO
CH3
HN
C CHN
O
CHCNH2
HN
C CHNH
O
CHCO
H
12
3 4 5
6
Cytosine Thyminein DNA
Uracilin RNA20
.1 T
he S
truc
ture
of
the
Nuc
leot
ide
Pyrimidines consist of a single 6-membered ring
Nucleotides
• A nucloetide is the repeating unit of the DNA or RNA polymer
• The nitrogen base is attached to – ribose (RNA) – deoxyribose (DNA)
• The sugar is phosphorylated at carbon 5'
NCCH
C
N
NC
CH
N
NH2
2-O3PO
OCH2
HOH
H
H
HH
base
deoxyribose sugar
phosphateester
20.1
The
Str
uctu
re o
f th
e N
ucle
otid
e
Deoxythymidine 5'-Monophosphate
Figure shows the linkages between
– The nitrogenous base thymidine and the 5-carbon sugar deoxyribose
– The deoxyribose and the phosphoryl group
C
C
N
C
C
NH
NH2
2-O3PO
OCH2
HOH
H
H
HH
CH3
O
Deoxythymidine 5'-monophosphate
dTMP
20.1
The
Str
uctu
re o
f th
e N
ucle
otid
e
The Specific Ribonucleotide, Adenosine Triphosphate
Schematic labels the different portions of the molecule indicating the change as sequential phosphoryl groups are added
20.1
The
Str
uctu
re o
f th
e N
ucle
otid
e
20.2 The Structure of DNA and RNA
• Nucleotides combine to form a chain or polymerize in a series of 3' to 5' phosphodiester bonds– The 5' phosphate on one unit
esterifies to the 3' OH on the adjacent unit
– The terminal 5' unit retains the phosphate
• Backbone of the polymer (in blue) is called the sugar-phosphate backbone because it is composed of alternating units of deoxyribose and phosphoryl groups
Segment of One DNA Chain
NCCH
C
N
NC
C
N
O
NH2-2
O3POO
CH2
H
O
H
H
HH
NC CH
N
O
CC
O
CH3
O P
O
OO
CH2
H
O
H
H
HH
NC CH
N
O
CHC
NH2
O P
O
OO
CH2
H
OH
H
H
HH
guanine
thymine
cytosine
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
5' end
3' end
3' carbon is linked to a 5' carbon via a
phosphodiester bond
Helical Structure of DNA
• DNA consists of two chains of nucleotides coiled around one another in a right-handed double helix– Sugar-phosphate backbones of the two
strands spiral around the outside of the helix like the handrails on a spiral staircase
– Nitrogenous bases extend into the center at right angles to the acids of the helix as if they are the steps of the spiral staircase
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
Hydrogen Bonding of the DNA Helix
• A noncovalent attraction aiding in maintaining the double helix structure is hydrogen bonding between base pairs – Adenine forms 2 H bonds with thymine A=T
– Cytosine forms 3 H bonds with guanine G=C
• This H bonding pattern is called base pairing• Diameter of the double helix is 2.0 nm
– Distance dictated by the dimensions of the purine-pyrimidine base pairs
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
Complementary DNA Strands
• The two DNA strands are complementary strands – The sequence of bases on one automatically
determine the sequence of bases on the other strand
• The chains run antiparallel– Only when the 2 strands are antiparallel can the base
pairs form the H bonds that hold them together
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
Insert Fig 24.4
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
Schematic Ribbon Diagram of DNA Double Helix
• 2 strands of DNA form a right-handed double helix
• Bases in opposite strands hydrogen bond according to the AT/GC rule
• 2 strands are antiparallel per their 5' to 3' directionality
• Per complete 360º turn of the helix there are 10 nucleotides
• One complete turn is 3.4 nm and one nucleotide is 0.34 nm
DNA Segment
Sugar-phosphate backbone
Hydrogen bondedbase pairs in thecore of the helix
Chain 1
Chain 2
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
Prokaryotic Chromosomes
• Chromosomes are pieces of DNA that contain the genetic instructions, or genes, of an organism
• Prokaryotes (single chromosome)– No true nucleus
– Chromosome is a circular DNA molecule that is supercoiled, meaning the helix is coiled on itself
– At approximately 40 sites, a complex of proteins is attached, forming a series of loops
– This structure is the nucleoid
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
Eukaryotic ChromosomesEukaryotes (number and size of chromosomes vary)
– True nucleus enclosed by a nuclear membrane
– Nucleosome which consists of a strand of DNA wrapped around a disk of histone proteins – DNA appears like beads on a string• String of beads then coils
into a larger structure called the 30 nm fiber
• With additional proteins next coiled in to a 200 nm fiber
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
Eukaryotic Chromosome Levels of Structure
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
RNA Structure
• Sugar-phosphate backbone for ribonucleotides is also linked by 3'-5' phosphodiester bonds– RNA molecules usually single-stranded– Ribose replaces deoxyribose– Uracil replaces thymine
• Base pairing between U and A and G and C can still occur– This H bonding results in portions of the
single-strand that become double-stranded
20.2
The
Str
uctu
re o
f D
NA
an
d R
NA
20.3 DNA Replication
• DNA must be replicated before a cell divides, so that each daughter cell inherits a copy of each gene– Cell missing a critical gene will die– Essential that the process of DNA replication
produces an absolutely accurate copy of the original genetic information
• Mistakes made in critical genes can result in lethal mutations
Structure to Function in DNA Replication
• Structure of the DNA molecule suggests the mechanism for accurate replication– An enzyme could “read” the nitrogenous bases on one
strand of a DNA molecule adding complementary bases to a newly synthesized strand
– Product of this strategy would be a new DNA molecule in which one strand is the original or parent strand, and the other is newly synthesized, a daughter strand
– This strategy is called semiconservative replication20.3
DN
A R
epli
cati
on
The Mechanism of DNA Replication
20.3
DN
A R
epli
cati
on
The Products of DNA Replication
• Semiconservative replication generates 2 new DNA helices– Each helix has 2 DNA
strands
– One strand is from the parental DNA (purple)
– The other strand is newly synthesized (blue)
20.3
DN
A R
epli
cati
on
Bacterial DNA Replication
• Bacterial chromosome is a circular molecule of DNA– Approximately 3 million nucleotides– DNA replication begins at a unique sequence, the
replication origin– Replication moves bidirectionally, 500 nucleotides per
second– Position where new nucleotides are added to the
growing daughter strand is the replication fork• As DNA synthesis moves bidirectionally, there are two
replication forks moving in opposite directions20.3
DN
A R
epli
cati
on
Bacterial DNA Replication20
.3 D
NA
Rep
lica
tion
The First Step in DNA Replication
First step is the separation of the strands1. Accomplished by helicase, which breaks the hydrogen
bonds between base pairs
2. Positive supercoiling results when hydrogen bonds are broken, this is relieved by topoisomerase
3. When supercoiling is relieved, single-strand binding protein binds to the separated strands to keep them apart
4. Primase catalyzes synthesis of a 10-12 base piece of RNA to “prime” the DNA replication20
.3 D
NA
Rep
lica
tion
Detail of DNA Replication20
.3 D
NA
Rep
lica
tion
Involved in first step
Involved in later stepsInvolved in later steps
DNA Polymerase Reaction
• After the first step is completed, DNA polymerase III “reads” the parental strand or template, catalyzing the polymerization of a complementary daughter strand
• In the polymerization reaction– A pyrophosphate group is released as a phosphoester
bond is formed between the 5'-phosphoryl group of the nucleotide being added, and the previous 3'-OH of the nucleotide in the newly synthesized daughter strand
– Based on the bond formed in the polymerization this is referred to a 5'- 3' synthesis20
.3 D
NA
Rep
lica
tion
DNA Polymerase Reaction20
.3 D
NA
Rep
lica
tion
Factors Influencing DNA Replication
• The two DNA strands being replicated are antiparallel to one another– DNA polymerase III can only catalyze in the 5'- 3'
direction– However, the replication fork moves in one direction
with both strands replicated simultaneously
• Small RNA primers are needed for a starting point of DNA replication
• RESULT: There are different mechanisms for replication of the two strands– The leading strand is replicated continuously– The opposite strand, the lagging strand, is replicated in
segments, or discontinuously
20.3
DN
A R
epli
cati
on
Leading Strand DNA Replication
• A single RNA primer is produced at the replication origin
• DNA polymerase III continuously catalyzes the addition of nucleotides in the 5'- 3' direction
20.3
DN
A R
epli
cati
on
Lagging Strand DNA Replication• Many RNA primers are produced as the replication
fork moves along the molecule• DNA polymerase III catalyzes the elongation of the
new strand in the 5'- 3' direction– As the new strand encounters a previously synthesized
new piece synthesis stops at that site– The process repeats with another primer made at a new
location of the replication fork• Final step is:
– The removal of the RNA primers – DNA polymerase I– Filling in the gaps– DNA polymerase I– Sealing the fragments into an intact strand of DNA –
DNA ligase
20.3
DN
A R
epli
cati
on
Detailed View of the Replication Fork20
.3 D
NA
Rep
lica
tion Lagging strand DNA synthesis is more easily
visualized here• DNA polymerase III reads:
– Discontinuously – In the opposite direction
Eukaryotic DNA Replication
• Discussion of prokaryotic DNA replication presents a complex picture
• DNA replication in eukaryotes is more complex still– One eukaryotic chromosome may be 100 times
larger than a bacterial chromosome– In eukaryotes, DNA replication begins at many
replication origins and moves bidirectionally along each chromosome20
.3 D
NA
Rep
lica
tion
20.4 Information Flow in Biological Systems
• Central Dogma tells us that “in cells the flow of genetic information contained in DNA is a one-way street that leads from DNA to RNA to protein”
• Transcription is the process by which a single-strand of DNA serves as a template for the synthesis of an RNA molecule– Think of making a COPY
• Translation converts the information from one language of nitrogenous bases to another of amino acids– Think of TRANSLATING into another language
Classes of RNA Molecules
• Messenger RNA (mRNA)– mRNA directs the amino acid sequence of proteins – A complimentary copy of a gene – It has the codon for an amino acid in a protein
• Ribosomal RNA (rRNA)– Structural and functional component of the ribosome – Forms ribosomes by reacting with proteins– 3 types in prokaryotes– 4 types in eukaryotes
• Transfer RNA (tRNA)– Transfers amino acids to the site of protein synthesis20
.4 I
nfor
mat
ion
Flo
w in
B
iolo
gica
l Sys
tem
s
tRNA• There is at least one tRNA for each amino acid to
be incorporated into a protein• tRNA is single-stranded with typically about 80
nucleotides• The overall structure is called a cloverleaf
– Intrachain hydrogen bonding (A=U and G=C) occurs to give:
• Regions called stems with an -helix• A type of L-shaped tertiary structure
– The 3'-OH group of the terminal nucleotide can covalently bind the amino acid
– 3 nucleotides at the base of the cloverleaf serve as the anticodon, which forms hydrogen bonds to a codon on mRNA20
.4 I
nfor
mat
ion
Flo
w in
B
iolo
gica
l Sys
tem
s
Structure of tRNA20
.4 I
nfor
mat
ion
Flo
w in
B
iolo
gica
l Sys
tem
s Free 3'-OH to bindthe amino acid
3 nucleotidesforming theanticodon
tRNATransfer RNA (tRNA) transfers the amino acid to the site of protein synthesis
Attachment tomRNA here
Amino AcidAttaches here
20.4
Inf
orm
atio
n F
low
in
Bio
logi
cal S
yste
ms
Transcription
• Transcription is catalyzed by RNA polymerase• Produces a copy of only 1 DNA strand • Process of transcription has 3 stages:
– Initiation binds RNA polymerase to the promoter region at the beginning of the gene
– Chain elongation then occurs forming a 3'-5' phosphodiester bond, generating a complementary copy
– Termination is the final step of transcription when the RNA polymerase releases the newly formed RNA molecule20
.4 I
nfor
mat
ion
Flo
w in
B
iolo
gica
l Sys
tem
s
Stages of Transcription20
.4 I
nfor
mat
ion
Flo
w in
B
iolo
gica
l Sys
tem
s
Initiation
Elongation
Termination
Post-transcriptional Processing of mRNA
• Prokaryotes release a mature mRNA at the end of termination for translation
• Eukaryote mRNA is a primary transcript which still must be processed in post-transcriptional modification, a three step process:– A 5' cap structure is added
• This structure is required for efficient translation of the final mRNA
– A 3' poly(A) tail (100 to 200 units) is added by poly(A) polymerase
• Poly(A) tail protects the 3' end of the mRNA from enzymatic digestion
• Prolongs the life of the mRNA 20.4
Inf
orm
atio
n F
low
in
Bio
logi
cal S
yste
ms
Addition of the 5'-Methylated Cap to mRNA
20.4
Inf
orm
atio
n F
low
in
Bio
logi
cal S
yste
ms
5'
Modification to the 5' end of the mRNA:
GuanosineMethylated at N-7
RNA Splicing
– RNA splicing is the removal of portions of the primary transcript that are not protein coding
• Bacterial chromosomes are continuous – all DNA sequence from the chromosome is found in the mRNA
• Eukaryotic chromosomes are discontinuous– There are extra DNA sequences within the genes
that do not encode any amino acid sequence called introns or intervening sequences
– Presence of introns makes direct translation to synthesize proteins impossible
• The introns are cut out and the exons (coding sequences) are spliced together
20.4
Inf
orm
atio
n F
low
in
Bio
logi
cal S
yste
ms
RNA Splicing Details
• RNA splicing must be very precise– Removing an incorrect number of nucleotides will
destroy the code for the protein– Signals mark the intron boundaries
• Spliceosomes help – Recognize intron-exon boundaries– Stabilize the splicing complex– They are composed of small nuclear
ribonucleoproteins (snRNPs, “snurps”)
20.4
Inf
orm
atio
n F
low
in
Bio
logi
cal S
yste
ms
Schematic Diagram of mRNA Splicing
20.4
Inf
orm
atio
n F
low
in
Bio
logi
cal S
yste
ms
20.5 The Genetic Code
The message on DNA that has been translated to mRNA:
1. Degenerate: more than one three base codon can code for the same amino acid
2. Specific: each codon specifies a particular amino acid
3. Nonoverlapping and commaless: • None of the bases are shared between consecutive codons
• No noncoding bases appear in the base sequence
4. Universal: all organisms use the same code
Genetic Code Details
• All 64 codons have meaning– 61 code for amino acids– Three code for the “stop” signal
• Multiple codes for an amino acid tend to have two bases in common– CUU, CUC, CUA, CUG code for leucine– Makes the code mutation resistant
• Codons are written in a 5' 3' sequence
20.5
The
Gen
etic
Cod
e
The Genetic Code20
.5 T
he G
enet
ic C
ode
Using The Genetic Code
1. CCU codes for?
2. CGA codes for?
3. UCA codes for?
proargser
20.5
The
Gen
etic
Cod
e
20.6 Protein Synthesis• Protein synthesis is called translation
– Carried out on ribosomes, complexes of • rRNA • Proteins
• Protein synthesis occurs in multiple places on one mRNA at a time – mRNA plus the multiple ribosomes are called a
polysome
• tRNA – Binds a specific amino acid aided by aminoacyl tRNA
synthetase– Recognizes the appropriate codon on the mRNA
Schematic of the Translation Process
20.6
Pro
tein
Syn
thes
is
Ribosomes
• Ribosomes are complexes of rRNA and proteins– Each ribosome is made up of 2 subunits
• Small ribosomal subunit contains 1 rRNA and 33 proteins
• Large ribosomal subunit contains 3 rRNA and about 49 proteins
– Many ribosomes on 1 mRNA comprise a polysome with many copies of the protein made simultaneously20
.6 P
rote
in S
ynth
esis
Structure of the Ribosome20
.6 P
rote
in S
ynth
esis
The Role of Transfer RNAMolecules that decode the information on the mRNA into the primary structure of the protein are the tRNA
– Requires two specific functions• Each tRNA must covalently bind one, and only one,
specific amino acid– Binding site for covalent attachment of the amino acid
at 3' end– Enzyme aminoacyl tRNA synthetase covalently links
the proper amino acid to the tRNA = aminoacyl tRNA• The tRNA must be able to recognize the appropriate
codon on the mRNA that calls for that amino acid– This process is mediated by the anticodon located at
the bottom of the tRNA cloverleaf– The anticodon is complementary to the codon on the
mRNA
20.6
Pro
tein
Syn
thes
is
Aminoacyl tRNA Synthetase20
.6 P
rote
in S
ynth
esis
The Process of Translation• Initiation
– Initiation factors (proteins), mRNA, initiator tRNA, and small and large ribosomes come together
– Ribosome has two sites to bind tRNA• P-site binds to the growing peptide• A-site binds the aminoacyl tRNA
• Chain elongation – a three step process1. An aminoacyl tRNA binds to A-site2. Peptide bond formation occurs catalyzed by peptidyl
transferase3. Translocation (movement) of ribosome down the
mRNA chain next to codon– Shifts the new peptidyl tRNA from the A-site to the P-site– Chain elongation requires hydrolysis of GTP to GDP
20.6
Pro
tein
Syn
thes
is
Insert Fig 24.19
20.6
Pro
tein
Syn
thes
is Protein Translation - Initiation
Insert Fig 24.19
20.6
Pro
tein
Syn
thes
is Protein Translation - Elongation
Termination of the Translation Process
• Termination– Upon finding a “stop” codon a release factor binds the
empty A-site– The bond between the last amino acid and peptidyl
tRNA is hydrolyzed releasing the protein
• The protein released may not be in its final form• Post-translational modification may occur before a
protein is fully functional – Cleavage– Association with other proteins– Bonding to carbohydrate or lipid groups20
.6 P
rote
in S
ynth
esis
Insert Fig 24.19
20.6
Pro
tein
Syn
thes
is Protein Translation - Termination
20.7 Mutation, Ultraviolet Light, and DNA Repair
• Mutations are mistakes introduced into the DNA sequence of an organism
• Mutations can be silent, that is, cause no change in the protein
• Many mutations have a negative effect on the health of the organism
• Many mutagens are also carcinogens and cause cancer– Chemicals causing a change in the DNA sequence
Mutation Classification
• Classified by the kind of change that occurs in the DNA:– Point: substitution of a single nucleotide for
another
– Deletion: one or more nucleotides are lost
– Insertion: one or more nucleotides are added
20.7
Mut
atio
n, U
ltra
viol
et
Lig
ht,
and
DN
A R
epai
r
UV Damage and DNA Repair
UV light causes covalent linkage of adjacent pyrimidine bases– Formation of a pyrimidine dimer on a DNA strand – Pyrimidine dimer formation can be used to kill bacteria
with UV exposure– Failure to repair this defect can lead to xeroderma
pigmentosum• People who suffer from this genetic skin disorder are very
sensitive to UV light and develop multiple skin cancers
20.7
Mut
atio
n, U
ltra
viol
et
Lig
ht,
and
DN
A R
epai
r
20.8 Recombinant DNA• Restriction enzymes are bacterial enzymes that cut
the backbone of DNA at specific nucleotide sequences
• Donor and plasmid (bacteria) DNA are cleaved by the same restriction enzyme
• Donor and plasmid DNA are mixed and donor fragment joins to a complimentary plasmid fragment due to hydrogen bonding
• Plasmid ring is restored using DNA ligase• Engineered plasmid (recombinant DNA) is
introduced to a bacterium to be reproduced
Restriction Enzymes
• Restriction enzymes are bacterial enzymes that “cut” the sugar-phosphate backbone of DNA at specific nucleotide sequences– An example of this type of enzyme is EcoRI, which cuts at:
– When the enzyme cuts the DNA it does so in a staggered fashion, cutting between the G and the first A of both strands, resulting in two DNA fragments
– The staggered termini are called sticky ends as they can reassociate with one another by hydrogen bonding
20.8
Rec
ombi
nant
DN
A
Common Restriction Enzymes and Their Recognition Sequences
• These enzymes are used to digest large DNA molecules into smaller fragments of specific size
• A restriction enzyme always cuts at the same site– DNA from a particular individual generates a
reproducible set of DNA fragments, which is useful for study of DNA from any source
20.8
Rec
ombi
nant
DN
A
Agarose Gel Electrophoresis• To study the DNA fragments produced by
restriction enzyme digestion one may employ agarose gel electrophoresis– Digested DNA sample is placed in a sample slot and
electric current is applied– Negative charge on the phosphoryl groups in the sugar-
phosphate backbone causes the DNA fragments to move through the gel, away from the negative electrode
• Smaller DNA fragments move faster resulting in a distribution of DNA fragments through the gel based on their size
20.8
Rec
ombi
nant
DN
A
Sample of Typical Agarose Gel DNA in wells DNA bands separated by size sample loaded after sample run
fragments separateby electric current
Hybridization• While agarose gel electrophoresis permits size
determination of DNA fragments, the identity of the gene in a DNA fragment is also very important
• Hybridization is a technique used to identify the presence of a gene on a particular DNA fragment– Based on the ability of complementary DNA
sequences to hybridize or hydrogen bond with each other
– RNA will also hybridize to DNA as well as to other RNA molecules20
.8 R
ecom
bina
nt D
NA
Southern Blot Hybridization20
.8 R
ecom
bina
nt D
NA
•Southern blotting hybridizes DNA fragments already separated on an agarose gel•The DNA fragments are transferred onto a special membrane filter which binds them very tightly
•The filter is exposed to a solution containing a radioactively-labeled DNA or RNA probe•This probe with a complementary sequence to the gene of interest will bind to any DNA fragment from the gel having the desired sequence
DNA Cloning Vectors• Using the techniques
described, a single gene can be:– Isolated– Placed in a cloning vector– Millions of copies made and
purified
• Cloning vector is a piece of DNA having its own replication origin, so it can be replicated inside a host cell– Phage vectors come from
bacterial viruses– Plasmid vectors are extra
pieces of circular DNA common to bacteria20
.8 R
ecom
bina
nt D
NA
Genetic EngineeringCloning a gene
• Select the gene to clone & rationale• Digest DNA sample from a known source
and the vector with a restriction enzyme• Mix the 2 DNA samples together so that the
sticky ends of sample and vector may hybridize and link those ends with DNA ligase
• Combine this DNA “package” with the bacterial cells to be transformed
• Grow bacteria containing our DNA package on an agar plate permitting only those bacteria with a gene from the vector to grow
• Transfer DNA from the surviving bacterial cells to a filter and hybridize with a probe to the gene being cloned
• Bacteria with the desired DNA can be detected, isolated, and grown
20.8
Rec
ombi
nant
DN
A
Some Medically Important Proteins That Are Produced by
Genetic Engineering
20.8
Rec
ombi
nant
DN
A
20.9 Polymerase Chain Reaction
• Polymerase chain reaction is a powerful technique that allows scientists to produce unlimited amounts of a gene of interest– The bacterium Thermus aquaticus produces a heat-
stable DNA polymerase (Taq polymerase), which drives this process
– Permits selection of just one gene of interest from among the 3 billion base pairs of DNA in the human genome
• Specificity is the primer, a short piece of single-stranded DNA that will hybridize specifically to the beginning of that gene of interest
PCR Three Step Reaction Sequence
• DNA is mixed with Taq polymerase and a primer DNA sequence designed for a specific gene, along with the four nucleotide triphosphates
• A thermocycler carefully regulates the temperature– Raises the temperature to 94-96oC for several minutes to
separate the DNA strands– Lowers the temperature to 50-56oC so that the primers
might hybridize to the target DNA present in the sample– Raises the temperature to 72oC to allow the Taq polymerase
to act – reading the template DNA strand and polymerizing a daughter strand extended from the supplied primer
• Repeating the cycle doubles the new DNA strands each cycle (12481632)20.9
Pol
ymer
ase
Cha
in
Rea
ctio
n
PCR Amplification of a Gene• After each PCR cycle the
amount of target DNA should double
• In theory, after 30 PCR cycles, there will be 1 billion times more DNA than at the start
• PCR is commonly used in:– Genetic screening
– Disease diagnosis
– Forensic detection of evidence20.9
Pol
ymer
ase
Cha
in
Rea
ctio
n
20.10 The Human Genome Project• The Human Genome Project was begun in
1990, as a multinational project that would:– Identify all of the genes in human DNA– Sequence the entire 3 billion nucleotide pairs of
the genome
• Original goal was to complete the project by 2005– Technological advances resulting in:
• A working draft in February 2001• Completion in April 2003
Genetic Strategies for Genome Analysis – Library
• In order to determine the DNA sequence of the human genome, genomic libraries were required
• Genomic library is a set of clones representing the entire genome– DNA sequence of each could then be
determined– This would not permit arrangement of these
sequence clones along the chromosome
20.1
0 T
he H
uman
Gen
ome
Pro
ject
Genetic Strategies for Genome Analysis – Walking
• Chromosome walking is an alternative technique providing both DNA sequence and a method for locating the DNA sequences next to it on the chromosome– This method requires overlapping clones– Libraries of clones were prepared using many different
restriction enzymes• Isolated DNA fragments are sequenced• Use sequence information to develop a probe for any
clones in that library which overlap the fragment sequenced• Process is continuous – scientists may work in both
directions at once until the entire sequence is cloned, mapped, and identified
20.1
0 T
he H
uman
Gen
ome
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DNA Sequencing Setup• The cloned piece of DNA is separated into its two
strands– Primer strand is also required to hybridize to the
template strand– Primer is the starting point for the addition of new
nucleotides
• The DNA to be sequenced is placed in four test tubes with all the enzymes and nucleotides necessary for DNA synthesis– Each test tube contains a small amount of one species
of dideoxynucleotide with a hydrogen at the 3' position– As this dideoxynucleotide is incorporated in the
growing chain, it acts as a chain terminator20.1
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DNA Sequencing Separation• Each of the four test tubes will contain a small
amount of only one of the dideoxynucleotides– The tube receiving dideoxyadenosine triphosphate will
produce DNA fragments– At some point in the synthesis a dideoxyadenosine
triphosphate will be added to the growing chain causing synthesis to stop
– This produces a family of fragments that all terminate with one and only one of the four dideoxynucleotides
• The reactions from each of the four tubes are separated on a long DNA sequencing gel
• DNA sequence can be determined directly from the gel
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DNA Sequencing Results
• Radioactive isotopes were the initial tag on the DNA fragments
• New technology labels with fluorescent dyes – a different color dye for each nucleotide– This permits all 4
reactions in one test tube
– All reaction products can be separated in one lane of a sequencing gel
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