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Chapter 20
Introduction to Molecular Genetics
Denniston
Topping
Caret
5th Edition
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
•
•
2.
Five-carbon sugar ring
•
•
3.
Purine
Pyrimidine
Ribose
Deoxyribose
Phosphoryl group
20.1 The Structure of the
Nucleotide
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 b-N-glycosidic
linkage joining the 1'-carbon
of the sugar and a nitrogen
atom of the base
20.1 The Structure of the
Nucleotide
Major Purine Bases
Nitrogenous 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
NH2
C
N1
HC
2
6
C
5
O
N
7
9
3 4C
N
Adenine
8 CH
N
H
C
HN
C
C
C
H2N
N
N
CH
N
H
Guanine
20.1 The Structure of the
Nucleotide
Major Pyrimidine Bases
Pyrimidines consist of a single 6-membered ring
O
O
NH2
C
CH
C
C
3
HN CH
N3 4 5CH HN C
2 1 6
C CH
C CH C CH
O N
O N
O N
H
H
H
Cytosine
Thymine
in DNA
Uracil
in RNA
20.1 The Structure of the
Nucleotide
Nucleotides
• A nucloetide is the
repeating unit of the
base
DNA or RNA
polymer
• The nitrogen base is phosphate
attached b to
ester
NH2
C
C N
N
C N
N
2O3PO CH2
O
• The sugar is
H
H
phosphorylated at
H
H
carbon 5'
deoxyribose sugar OH H
– ribose (RNA)
– deoxyribose (DNA)
HC
CH
20.1 The Structure of the
Nucleotide
Deoxythymidine 5'-Monophosphate
Figure shows the
linkages between
– The nitrogenous
base thymidine
and the 5-carbon
sugar
deoxyribose
– The deoxyribose
and the
phosphoryl group
NH2
C CH3
C
HN
C
N
O
2O3PO CH2
O
H
H
H
H
OH H
C
Deoxythymidine
5'-monophosphate
dTMP
20.1 The Structure of the
Nucleotide
The Specific Ribonucleotide,
Adenosine Triphosphate
Schematic labels the different portions of the
molecule indicating the change as sequential
phosphoryl groups are added
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 sugarphosphate backbone because
it is composed of alternating
units of deoxyribose and
phosphoryl groups
20.2 The Structure of DNA
and RNA
Segment of One DNA Chain
5' end
N
O
C
C N
C N
C
H2N
N
-2
O3PO CH2
O
H
H
H
H
H
O
guanine
CH
N
C
O
C
O
N
O P O CH2
O
O
H
H
H
H
linked
H
O
3' carbon is
to a 5' carbon via a
phosphodiester
bond
CH3
C
CH
NH2
C
N
CH
C
CH
O
N
O P O CH2
O
O
H
H
H
H
OH H
3' end
thymine
cytosine
20.2 The Structure of DNA
and RNA
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 Structure of DNA
and RNA
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 purinepyrimidine base pairs
20.2 The Structure of DNA
and RNA
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 Structure of DNA
and RNA
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
Fig
AT/GC Insert
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
24.4
20.2 The Structure of DNA
and RNA
DNA Segment
Sugar-phosphate
backbone
Chain 2
Chain 1
Hydrogen bonded
base pairs in the
core of the helix
20.2 The Structure of DNA
and RNA
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 Structure of DNA
and RNA
Eukaryotic Chromosomes
Eukaryotes (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 Structure of DNA
and RNA
Eukaryotic Chromosome Levels
of Structure
20.2 The Structure of DNA
and RNA
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
20.3 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
20.3 DNA Replication
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 replication
20.3 DNA Replication
The Mechanism of DNA
Replication
20.3 DNA Replication
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 DNA Replication
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 directions
20.3 DNA Replication
Bacterial DNA Replication
20.3 DNA Replication
The First Step in DNA
Replication
First step is the separation of the strands
1. 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 replication
20.3 DNA Replication
Detail of DNA Replication
Involved in first step
Involved in later steps
20.3 DNA Replication
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' synthesis
20.3 DNA Replication
DNA Polymerase Reaction
20.3 DNA Replication
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 DNA Replication
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 DNA Replication
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 DNA Replication
Detailed View of the Replication Fork
Lagging strand DNA synthesis is more easily
visualized here
• DNA polymerase III reads:
– Discontinuously
– In the opposite direction
20.3 DNA Replication
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 chromosome
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
20.4 Information Flow in
Biological Systems
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 synthesis
20.4 Information Flow in
Biological Systems
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 a-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
mRNA
20.4 Information Flow in
Biological Systems
Structure of tRNA
Free 3'-OH to bind
the amino acid
3 nucleotides
forming the
anticodon
20.4 Information Flow in
Biological Systems
tRNA
Transfer RNA (tRNA) transfers the amino acid to the
site of protein synthesis
Attachment to
mRNA here
Amino Acid
Attaches here
20.4 Information Flow in
Biological Systems
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 molecule
20.4 Information Flow in
Biological Systems
Stages of Transcription
Initiation
Elongation
Termination
20.4 Information Flow in
Biological Systems
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 Information Flow in
Biological Systems
Addition of the 5'-Methylated Cap
to mRNA
5'
Modification to the 5'
end of the mRNA:
Guanosine
Methylated at N-7
20.4 Information Flow in
Biological Systems
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 Information Flow in
Biological Systems
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 Information Flow in
Biological Systems
Schematic Diagram of mRNA
Splicing
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
20.5 The Genetic 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 Genetic Code
The Genetic Code
20.5 The Genetic Code
Using The Genetic Code
1. CCU codes for? pro
2. CGA codes for? arg
3. UCA codes for? ser
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
20.6 Protein Synthesis
Schematic of the Translation
Process
20.6 Protein Synthesis
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
simultaneously
20.6 Protein Synthesis
Structure of the Ribosome
20.6 Protein Synthesis
The Role of Transfer RNA
Molecules 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 Protein Synthesis
Aminoacyl tRNA Synthetase
20.6 Protein Synthesis
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 process
1. An aminoacyl tRNA binds to A-site
2. Peptide bond formation occurs catalyzed by peptidyl
transferase
3. 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 Protein Synthesis
Protein Translation - Initiation
Insert Fig 24.19
20.6 Protein Synthesis
Protein Translation - Elongation
Insert Fig 24.19
20.6 Protein Synthesis
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 groups
20.6 Protein Synthesis
Protein Translation - Termination
Insert Fig 24.19
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
20.7 Mutation, Ultraviolet
Light, and DNA Repair
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 Mutation, Ultraviolet
Light, and DNA Repair
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
20.8 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
20.8 Recombinant DNA
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 Recombinant DNA
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 Recombinant DNA
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 sugarphosphate 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
Sample of Typical Agarose Gel
DNA in wells
sample loaded
DNA bands separated by size
after sample run
fragments separate
by electric current
20.8 Recombinant DNA
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 molecules
20.8 Recombinant DNA
Southern Blot Hybridization
•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
20.8 Recombinant DNA
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 bacteria
20.8 Recombinant DNA
Genetic Engineering
•
•
•
•
•
•
•
Cloning 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 Recombinant DNA
Some Medically Important
Proteins That Are Produced by
Genetic Engineering
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 heatstable 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 singlestranded DNA that will hybridize specifically to
the beginning of that gene of interest
20.9 Polymerase Chain
Reaction
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 (12481632)
20.9 Polymerase Chain
Reaction
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 evidence
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
20.10 The Human Genome
Project
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
20.10 The Human Genome
Project
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
20.10 The Human Genome
Project
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 terminator
20.10 The Human Genome
Project
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
20.10 The Human Genome
Project
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