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

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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

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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

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of

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Nuc

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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

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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

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The Specific Ribonucleotide, Adenosine Triphosphate

Schematic labels the different portions of the molecule indicating the change as sequential phosphoryl groups are added

20.1

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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

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NA

an

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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

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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

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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

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an

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Insert Fig 24.4

20.2

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an

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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

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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

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an

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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

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Eukaryotic Chromosome Levels of Structure

20.2

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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

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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

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The Mechanism of DNA Replication

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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)

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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

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Bacterial DNA Replication20

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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

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Rep

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Detail of DNA Replication20

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Rep

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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

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Rep

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DNA Polymerase Reaction20

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Rep

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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

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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

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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

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Detailed View of the Replication Fork20

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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

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Rep

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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

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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

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Structure of tRNA20

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Flo

w in

B

iolo

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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

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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

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Stages of Transcription20

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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

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Addition of the 5'-Methylated Cap to mRNA

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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

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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”)

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Schematic Diagram of mRNA Splicing

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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

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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

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Structure of the Ribosome20

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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

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is

Aminoacyl tRNA Synthetase20

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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

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tein

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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

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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

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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

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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

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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

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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

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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

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Southern Blot Hybridization20

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•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

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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

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Some Medically Important Proteins That Are Produced by

Genetic Engineering

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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

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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

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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

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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

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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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