1

Recombinant DNA Technology

Stephen B. Gruber, MD, PhD

Division of Molecular Medicine and Genetics

November 4, 2002

Learning Objectives

• Know the basics of gene structure, function and regulation.

• Be familiar with the basic methods of molecular genetics.

• Understand the meaning of DNA sequence and amino acidpolymorphisms.

• Know how DNA sequence analysis is performed and befamiliar with methods of screening for differences.

• Have a general understanding of methods for gene transferinto tissue culture cells and the power of transgenictechnologies.

2

Learning Objectives (1)

• Know the basics of gene structure, function and regulation.

• Be familiar with the basic methods of molecular genetics.

• Understand the meaning of DNA sequence and amino acidpolymorphisms.

• Know how DNA sequence analysis is performed and befamiliar with methods of screening for differences.

• Have a general understanding of methods for gene transferinto tissue culture cells and the power of transgenictechnologies.

Chromosomes, DNA, and Genes

CellNucleus

Chromosomes

Gene

Protein

Adapted from Understanding Gene Testing, NIH, 1995

3

Genetic Code

A codon is made of 3 base pairs

64 codons total

1 codon (AUG) encodesmethionine and starts

translation of all proteins

3 codons stopprotein

translation

61 codons encode 20amino acids

(redundant code)

U A A

A U G

Met

G C A

Ala

DNA Transcription andTranslation

mRNA

Ribosome

Growingchain of

amino acids

Protein

Nuclearmembrane Cell membrane

DNA

Adapted from Understanding Gene Testing, NIH, 1995

4

5' end

Promoter

RNA transcriptionstart site

3' end

Gene Structure

Stop site

Intron Exon 2 IntronExon 1 Exon 3

Splice sites

Exon 2Exon 1 Exon 3

mRNA

RNA Processing

Translation

Protein

DNA

PrimarymRNA

MaturemRNA

Processing

Transcription

Exon Intron Exon Intron Exon

GU AG

5

Learning Objectives (2)

• Know the basics of gene structure, function and regulation.

• Be familiar with the basic methods of molecular genetics.– nucleic acid hybridization– Southern (DNA) and northern (RNA) blotting– PCR– DNA sequencing– basic steps involved in constructing & screening a cDNA library

• Understand the meaning of DNA sequence and amino acidpolymorphisms.

• DNA sequence analysis

• Transgenic technologies

from Textbook: 5.4

1944DNA is the

genetic material

1949Abnl Hemoglobin

in sickle cell anemia

1953Double helix

1956Glu 6 Val insickle hemoglobin

1966Completion of the

genetic code

1970First restriction

enzyme

1972Recombinant

plasmids

1975Southernblotting

1981Transgenic mice

1983Huntington

Disease genemapped

1985PCR

1986Positional cloning

(CGD, musculardystrophy,

retinoblastoma

1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000

1987Knockout

mice

1989Positional cloningwithout deletion (CF)

1990First NIH-approvedgene therapyexperiment

1996Complete yeastgenome sequence

19951st completebacterialgenome sequence

2001Draft human

genome sequence

6

Preparing DNA for Analysis

Blood sample Centrifuge andextract DNA fromwhite blood cells

DNA for analysis

SINGLE-STRANDEDDNA PROBESFOR GENE A

MIXTURE OF SINGLE-STRANDEDDNA MOLECULES

+ B

B B

A

A

C

CC

D

D

D

E

EE

F

F F

ONLY A FORMS A STABLEDOUBLE-STRANDED COMPLEXES

A, C, E ALL FORMSTABLE COMPLEXES

STRINGENT HYBRIDIZATION REDUCED-STRINGENCY HYBRIDIZATION

A

Textbook: Figure 5.8

7

Electrophoresis of DNA

Voltage

+

DNA fragments loaded into wells

Path of migration

DNA fragmentsseparate by size

and charge

_

Electrophoresis

Restriction enzymedigestion

Principle of a Southern blothybridize labeled probe to fragment of DNA

Add radio-labelednormal DNA

probes

8

Polymerase Chain Reaction(PCR)

Isolate anddenature DNA

Anneal andextend primers

Repeat asnecessary

Amplifiedsegments

Sequence to beamplified

DNA Sequencing

9

5'3'

T G T T

C T G A C T T C G A C A A

SINGLE-STRANDED DNAOF UNKNOWN SEQUENCE

RADIOACTIVELY LABELEDPRIMER

O CH O2

H H

H H

HH

DIDEOXYNUCLEOTIDE (ddNTP)

DNA POLYMERASE I

dATP

dGTP

dCTP

dTTP

ddATP ddCTP ddTTP ddGTP

C T G A C T T C G A C A A

ddG

REACTIONMIXTURES

dd

AT

P

READ SEQUENCE OF ORIGINALSINGLE-STRANDED DNA(COMPLEMENT OF PRIMER-GENERATED SEQUENCE LADDER)

GELELECTROPHORESIS

AUTORADIOGRAPHYTO DETECT

RADIOACTIVE BANDS

3'5'

BASE

dd

CT

P

dd

TT

P

dd

GT

PLARGER

FRAGMENTS

SMALLERFRAGMENTS

C

T

G

A

CT

T

C

G

ddG

ddG

PRODUCTS IN ddGTP REACTION

PP P

Textbook: Figure 5.17

DNA Sequencing

ATC TTA GAG TGT CCC ATC TTA GTG TCC C

Start

A T C G

Normal Mutant (185delAG)

AG

A T C G

delA

Start

delG

10

Learning Objectives (3)

• Know the basics of gene structure, function and regulation.

• Be familiar with the basic methods of molecular genetics.– nucleic acid hybridization– Southern (DNA) and northern (RNA) blotting– PCR and gel electrophoresis– DNA sequencing– basic steps involved in constructing & screening a cDNA library

• Understand the meaning of DNA sequence and amino acidpolymorphisms.

• DNA sequence analysis

• Transgenic technologies

Polymorphisms and Mutations• Sequence variation-- differences among individuals

(DNA, amino acid)– > 0.01 = polymorphism– < 0.01 = rare variant

• Mutation-- any change in DNA sequence– Silent vs. amino acid substitution vs. other– neutral vs. disease-causing

• Common but incorrect usage:

“mutation vs. polymorphism”

• balanced polymorphism= disease + polymorphism

11

Learning Objectives (3)(continued)

• Understand the meaning and significance of DNAsequence and amino acid polymorphisms.

• Understand the various types of DNA sequencepolymorphisms.– RFLPs (Restriction Fragment Length Polymorphism)

– VNTRs (Variable Number Tandem Repeat)

– SSRs (Simple Sequence Repeat; also STR [Short/SimpleTandem Repeat]))

– SNPs (Single Nucleotide Polymorphism)

Textbook: Figure 5.19

12

Learning Objectives (3)(continued)

• Understand the meaning and significance of DNAsequence and amino acid polymorphisms.

• Understand the various types of DNA sequencepolymorphisms.– RFLPs (Restriction Fragment Length Polymorphism)

– VNTRs (Variable Number Tandem Repeat)

– SSRs (Simple Sequence Repeat; also STR [Short/SimpleTandem Repeat]))

– SNPs (Single Nucleotide Polymorphism)

Disease-Associated Mutations Alter Protein Function

Functional protein Nonfunctional ormissing protein

13

P1 P2(TCTA)10

(TCTA)11

(TCTA)12

(TCTA)13

(TCTA)14

(TCTA)15

A

B

C

D

E

F

AB CD EF AF CE

15

14

13

1211

10

Textbook: Figure 5.22

SNP (coding sequence)

NormalmRNA

Protein

A U G

Met

A A G

Lys

U U U

Phe

G G C

Gly

G C A

Ala

U U G

Leu

A A

Gln

C

Silent DNA sequence polymorphism

Sequencevariant

mRNA

ProteinA U G

Met

A A G

Lys

U U U

Phe

G G U

Gly

G C A

Ala

U U G

Leu

A A

Gln

C

G

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Disease-Associated Mutations

A mutation is a change in the normal base pair sequence

Commonly used to define DNA sequence changesthat alter protein function

PolymorphismDNA sequence changes that do not alter

protein function (common definition, not technically correct)

Functional protein Functional protein

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Polymorphism• Variation in population

– phenotype– genotype (DNA sequence polymorphism)

• Variant allele > 1%

“Normal”

Disease

< 1% > 1%

Rare or “private”polymorphism

polymorphism

Common usage:

disease ??Factor V R506Q: thrombosis, 3% allele frequency

THE BIG RED DOG RAN OUT.

THE BIG RAD DOG RAN OUT.

THE BIG RED.

THE BRE DDO GRA.

THE BIG RED ZDO GRA.

Mutations

Normal

Missense

Nonsense

Frameshift (deletion)

Frameshift (insertion)

Point mutation: a change in a single base pair

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Silent Sequence Variants

NormalmRNA

Protein

A U G

Met

A A G

Lys

U U U

Phe

G G C

Gly

G C A

Ala

U U G

Leu

A A

Gln

C

Sequence variant: a base pair change that does not change theamino acid sequence (a type of polymorphism)

Sequencevariant

mRNA

Protein

Adapted from Campbell NA (ed). Biology, 2nd ed, 1990

A U G

Met

A A G

Lys

U U U

Phe

G G U

Gly

G C A

Ala

U U G

Leu

A A

Gln

C

G

Missense Mutations

Missense

Missense: changes to a codon for another amino acid(can be harmful mutation or neutral polymorphism)

mRNA

Protein

NormalmRNA

Protein

A U G

Met

A A G

Lys

U U U

Phe

G G C

Gly

G C A

Ala

U U G

Leu

A U G

Met

A A G

Lys

U U U

Phe

A G C

Ser

G C A

Ala

U U G

Leu

A A

Gln

C

A A

Gln

C

Adapted from Campbell NA (ed). Biology, 2nd ed, 1990

17

Nonsense Mutations

Nonsense: change from an amino acid codon to a stopcodon, producing a shortened protein

Nonsense

mRNA

Protein

NormalmRNA

Protein

A U G

Met

A A G

Lys

U U U

Phe

G G C

Gly

G C A

Ala

U U G

Leu

A U G

Met

U A G U U U G G C G C A U U G

A A

Gln

C

A AC

Adapted from Campbell NA (ed). Biology, 2nd ed, 1990

Frameshift Mutations

Frameshift U G C A AA U G

Met

A A G

Lys

G C G

Ala

C A UU U

U

G

Leu

Frameshift: insertion or deletion of base pairs, producing a stopcodon downstream and (usually) shortened protein

mRNA

Protein

NormalmRNA

Protein

A U G

Met

A A G

Lys

U U U

Phe

G G C

Gly

G C A

Ala

U U G

Leu

A A

Gln

C

Adapted from Campbell NA (ed). Biology, 2nd ed, 1990

18

Splice-Site Mutations

Exon 1 Intron Exon 2 Intron Exon 3

Exon 1 Exon 3Altered mRNA

Splice-site mutation: a change that results in altered RNA sequence

Exon 2

Other Types of Mutations

• Mutations in regulatory regions of the gene

• Large deletions or insertions

• Chromosomal translocations or inversions

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Types of Mutations

• Point Mutations– Silent– Missense– Nonsense– (frameshift)

• Deletion/Insertion– small– large

• Rearrangement

• Transcription

• RNA Processing– splicing– poly A– RNA stability

• Protein level– processing– stability– altered function

• gain• loss• new

Learning Objectives (4)

• Know the basics of gene structure, function and regulation.

• Be familiar with the basic methods of molecular genetics.

• Understand the meaning of DNA sequence and amino acidpolymorphisms.

• Know how DNA sequence analysis is performed and befamiliar with methods of screening for differences. – SSCP– DGGE– CSGE– ASO– Chip technology

• methods for gene transfer and the power of transgenics

20

Tests to Detect Unknown Mutations

• Used when a specific mutation has not beenpreviously identified in a family

• DNA sequencing is most informative method

• Simpler scanning tests also may be used, usuallyfollowed by limited sequencing to characterizethe specific mutation

Single Strand ConformationalPolymorphism (SSCP)

DNA

Gel

Normal Mutated

mutation

• DNA is denatured intosingle strands

• Single strands fold; shapeis altered by mutations

• Mobility of mutant andnormal strands differ ingel

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Evaluating SSCP

Pros

• Rapid, simple, and widelyavailable for many genes

• Detects 60%−95% ofmutations in short DNAstrands

Cons

• Subsequent DNA sequencingneeded to characterize mutation

• Sensitivity drops with longerDNA sequences

Denaturing Gradient GelElectrophoresis (DGGE)

• DNA denatured into singlestrands

• Single strands reanneal intonormal and mutanthomoduplexes andheteroduplexes

• Hetero- and homoduplexesdenature at different points ingradient gel

DNA

Denaturing gradient gel

Normal Mutated

22

Denaturing Gradient Gel

1 normal homoduplex band2 heteroduplex bands1 mutant homoduplex band

BRCA1 mutation carrier

Evaluating DGGE

Pros• Highly sensitive (>90%)

• Better resolution than SSCP

Cons• Not efficient for

analyzing large DNAfragments

• Subsequent DNAsequencing needed tocharacterize mutation

• Labor-intensive set-up

23

Heteroduplex Analysis (CSGE)

Normal band

Mutated bands

Single-strand DNACold

Reannealed DNA

Amplify anddenature

DNA

Evaluating HeteroduplexAnalysis

Pros

• >90% sensitivity

• Rapid, simple assay

• Easily automated for highthroughput use

Cons

• Subsequent sequencingneeded to characterizemutation

24

Tests to Search for KnownMutations

• Used when a specific mutation is known or suspected tooccur in a family

• Methods focus on detection of one or a few specificmutations (eg, “Ashkenazi Jewish panel”)

• Methods include ASO, CSGE, restriction site digestion,others

Add radio-labelednormal DNA

probes

Amplify DNA and hybridizeto membranes

Allele Specific Oligonucleotide(ASO) Hybridization

Add knownmutant DNA

probes

Patients

#1 #2 #3

#1 #2 #3

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Evaluating ASO Analysis

Pros

• Sensitive method to detectknown mutations

• Panels of ASO probes usefulto detect common mutations

Cons

• Each ASO probe detectsonly one specific sequence

• Most useful for smallsequence changes

Principle of Microarray (Chip)Assay

Synthetic DNA probes

Prehybridization Posthybridization

Probes withhybridized DNA

26

Mutation vs. Silent Sequence Variation

• Obvious disruption of gene– large deletion or rearrangement– frameshift– nonsense mutation

• Functional analysis of gene product– expression of recombinant protein– transgenic mice

• New mutation by phenotype and genotype

X

Learning Objectives (5)

• Know the basics of gene structure, function and regulation.

• Be familiar with the basic methods of molecular genetics.

• Understand the meaning of DNA sequence and amino acidpolymorphisms.

• Know how DNA sequence analysis is performed and befamiliar with methods of screening for differences.

• Have a general understanding of methods for gene transferinto tissue culture cells and the power of transgenictechnologies.

27

REMOVE FERTILIZED OOCYTES FROM OVULATING MOUSE IMMEDIATELY

AFTER FERTILIZATION

REMOVE BLASTOCYSTS FROM PREGNANT MOUSE FOUR DAYS AFTER OVULATION

FEMALE PRONUCLEUSHOLDING PIPETTE

INJECTION NEEDLE IMPALING MALE PRONUCLEUS OF OOCYTE AND INJECTING DNA

OOCYTE

REIMPLANT SEVERAL OOCYTES IN FOSTER MOTHER

REIMPLANT SEVERAL BLASTOCYSTS IN FOSTER MOTHER

BIRTH

BIRTH

BIRTH

A

C

B

D

SOUTHERN BLOT OF TAIL DNA NORTHERN BLOT

BREEDING

A B C D

C

A

C

B

D

A B C D

SOUTHERN BLOT OF TAIL DNAA B C D

CULTURED ES CELLS WITH TARGETED GENE

ALTERATION

INJECT ES CELLS INTO BLASTOCYST

+

NORMAL GENE

ALTERED GENE

Summary

• Gene structure helps us understand where to look for errors.

• PCR and gel electrophoresis essential for diagnostic tests.

• DNA polymorphisms are best defined by frequency.

• Screening for DNA sequence differences is performed bydirect sequencing or other techniques that are selected basedon whether the mutation is known or unknown.

• Introduction to gene transfer provides a framework forlearning about gene therapy and methods for recombinantdrug development.