recombinant dna technology stephen b. gruber, md, phd division of molecular medicine and genetics...

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 acid

polymorphisms. • Know how DNA sequence analysis is performed and be

familiar with methods of screening for differences. • Have a general understanding of methods for gene transfer

into tissue culture cells and the power of transgenic technologies.

Learning Objectives (1)





Chromosomes, DNA, and Genes

CellCellNucleusNucleus

ChromosomesChromosomes

Gene

ProteinProtein

Adapted from Adapted from Understanding Gene TestingUnderstanding Gene Testing, NIH, 1995, NIH, 1995

Genetic Code

A codon is made of 3 base pairs

64 codons total

1 codon (AUG) encodes 1 codon (AUG) encodes methionine methionine andand starts starts

translation of all proteinstranslation of all proteins

3 codons stop 3 codons stop protein protein

translationtranslation

61 codons encode 20 61 codons encode 20 amino acidsamino acids

(redundant code)(redundant code)

U A A

A U G

Met

G C A

Ala

DNA Transcription and Translation

mRNAmRNA

RibosomeRibosome

Growing Growing chain of chain of

amino acidsamino acids

ProteinProtein

Nuclear Nuclear membranemembrane Cell membraneCell membrane

DNADNA

Adapted from Adapted from Understanding Gene TestingUnderstanding Gene Testing, NIH, 1995, NIH, 1995

5' end 5' end

PromoterPromoter

RNA transcription RNA transcription start sitestart site

3' end 3' end

Gene Structure

Stop siteStop site

Intron Exon 2 IntronExon 1 Exon 3

Splice sitesSplice sites

Exon 2Exon 1 Exon 3

mRNAmRNA

RNA Processing

TranslationTranslation

ProteinProtein

DNADNA

Primary Primary mRNAmRNA

Mature Mature mRNAmRNA

ProcessingProcessing

TranscriptionTranscription

ExonExon IntronIntron ExonExon IntronIntron ExonExon

GU AG


• 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 acid polymorphisms.

• 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 in sickle hemoglobin

1966Completion of the

genetic code

1970First restriction

enzyme

1972Recombinant

plasmids

1975Southern blotting

1981Transgenic mice

1983Huntington

Disease gene mapped

1985PCR

1986Positional cloning

(CGD, muscular dystrophy,

retinoblastoma

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

1987Knockout

mice

1989Positional cloning without deletion (CF)

1990First NIH-approved gene therapy experiment

1996Complete yeast genome sequence

19951st complete bacterialgenome sequence

2001Draft human

genome sequence

Preparing DNA for Analysis

Blood sampleBlood sample Centrifuge and Centrifuge and extract DNA from extract DNA from white blood cellswhite blood cells

DNA for analysisDNA 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

Electrophoresis of DNA

VoltageVoltage

++

DNA fragments loaded into wellsDNA fragments loaded into wells

Path of migrationPath of migration

DNA fragments DNA fragments separate by size separate by size

and chargeand charge

__

ElectrophoresisElectrophoresis

Restriction enzyme Restriction enzyme digestiondigestion

Principle of a Southern blothybridize labeled probe to fragment of DNA

Add radio-labeled Add radio-labeled normal DNA normal DNA

probesprobes

Polymerase Chain Reaction (PCR)

Isolate and Isolate and denature DNAdenature DNA

Anneal and Anneal and extend primersextend primers

Repeat as Repeat as necessarynecessary

Amplified Amplified segmentssegments

Sequence to be Sequence to be amplifiedamplified

DNA Sequencing

5'3'

T G T T

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

SINGLE-STRANDED DNAO F UNKNO WN SEQ UENCE

RADIO ACTIVELY LABELEDPR IMER

O CH O2

H H

H H

HH

DIDEOXYNUC LEO TID E (ddNTP)

DNA POLYMERASE I

dATP

dG TP

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 O RIG INALSINGLE-STRANDED DNA(COM PLEMENT O F PR IMER-G ENERATED SEQ UENCE LADDER)

G ELELECTROPHO RESIS

AUTO RADIO G RAPH YTO DETECT

RADIO ACTIVE BANDS

3'5'

BASE

dd

CT

P

dd

TT

P

dd

GT

P

LARG ERFRAG MEN TS

SM ALLERFRAG MEN TS

C

T

G

A

CT

T

C

G

ddG

ddG

PR ODUCTS IN ddGTP REACTION

PP P


DNA Sequencing

ATC TTA GAG TGT CCCATC TTA GAG TGT CCC ATC TTA GTG TCC CATC TTA GTG TCC C

StartStart

AA TT CC GG

NormalNormal Mutant (185delAG)Mutant (185delAG)

AGAG

AA TT CC GG

delAdelA

StartStart

delGdelG


• 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 acid polymorphisms.

• 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

Learning Objectives (3) (continued)

• Understand the meaning and significance of DNA sequence and amino acid polymorphisms.

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

– VNTRs (Variable Number Tandem Repeat)

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

– SNPs (Single Nucleotide Polymorphism)

Disease-Associated Mutations Alter Protein Function

Functional proteinFunctional protein Nonfunctional or Nonfunctional or missing proteinmissing protein

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


SNP (coding sequence)

NormalNormalmRNAmRNA

ProteinProtein

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

Sequence Sequence variantvariant

mRNAmRNA

ProteinProteinA 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

Disease-Associated Mutations

A mutation is a change in the normal base pair sequence

Commonly used to define DNA sequence changes Commonly used to define DNA sequence changes that alter protein functionthat alter protein function

PolymorphismDNA sequence changes that do DNA sequence changes that do notnot alter alter

protein function (common definition, protein function (common definition, not technically correctnot technically correct) )

Functional proteinFunctional protein Functional proteinFunctional protein

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 pairPoint mutation: a change in a single base pair

Silent Sequence Variants


ProteinProtein

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

Sequence Sequence variantvariant

mRNAmRNA

ProteinProtein

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

MissenseMissense

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

mRNAmRNA

ProteinProtein


ProteinProtein

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


Nonsense Mutations

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

NonsenseNonsense

mRNAmRNA

ProteinProtein


ProteinProtein

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


Frameshift Mutations

FrameshiftFrameshift 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 stop Frameshift: insertion or deletion of base pairs, producing a stop codon downstream and (usually) shortened proteincodon downstream and (usually) shortened protein

mRNAmRNA

ProteinProtein


ProteinProtein

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


Splice-Site Mutations

Exon 1Exon 1 IntronIntron Exon 2Exon 2 IntronIntron Exon 3Exon 3

Exon 1Exon 1 Exon 3Exon 3Altered mRNAAltered mRNA

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

Exon 2Exon 2

Other Types of Mutations

• Mutations in regulatory regions of the geneMutations in regulatory regions of the gene

• Large deletions or insertionsLarge deletions or insertions

• Chromosomal translocations or inversionsChromosomal translocations or inversions

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




familiar with methods of screening for differences. – SSCP – DGGE – CSGE– ASO– Chip technology

• methods for gene transfer and the power of transgenics

Tests to Detect Unknown Mutations

• Used when a specific mutation has not been previously identified in a family

• DNA sequencing is most informative method

• Simpler scanning tests also may be used, usually followed by limited sequencing to characterize the specific mutation

Single Strand Conformational Polymorphism (SSCP)

DNADNA

GelGel

NormalNormal MutatedMutated

mutationmutation

• DNA is denatured into single strands

• Single strands fold; shape is altered by mutations

• Mobility of mutant and normal strands differ in gel

Evaluating SSCP

Pros

• Rapid, simple, and widely available for many genes

• Detects 60%95% of mutations in short DNA strands

Cons

• Subsequent DNA sequencing needed to characterize mutation

• Sensitivity drops with longer DNA sequences

Denaturing Gradient Gel Electrophoresis (DGGE)

• DNA denatured into single strands

• Single strands reanneal into normal and mutant homoduplexes and heteroduplexes

• Hetero- and homoduplexes denature at different points in gradient gel

DNADNA

Denaturing gradient gelDenaturing gradient gel

NormalNormal MutatedMutated

Denaturing Gradient Gel

1 normal homoduplex band1 normal homoduplex band2 heteroduplex bands2 heteroduplex bands1 mutant homoduplex band1 mutant homoduplex band

BRCA1BRCA1 mutation carrier mutation carrier

Evaluating DGGE

Pros• Highly sensitive (>90%)

• Better resolution than SSCP

Cons• Not efficient for

analyzing large DNA fragments

• Subsequent DNA sequencing needed to characterize mutation

• Labor-intensive set-up

Heteroduplex Analysis (CSGE)

Normal bandNormal band

Mutated bandsMutated bands

Single-strand DNASingle-strand DNAColdCold

Reannealed DNAReannealed DNA

Amplify and Amplify and denature denature

DNADNA

Evaluating Heteroduplex Analysis

Pros

• >90% sensitivity

• Rapid, simple assay

• Easily automated for high throughput use

Cons

• Subsequent sequencing needed to characterize mutation

Tests to Search for Known Mutations

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

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

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

Add radio-labeled Add radio-labeled normal DNA normal DNA

probesprobes

Amplify DNA and hybridize Amplify DNA and hybridize to membranesto membranes

Allele Specific Oligonucleotide(ASO) Hybridization

Add known Add known mutant DNA mutant DNA

probesprobes

PatientsPatients

#1#1 #2#2 #3#3

#1#1 #2#2 #3#3

Evaluating ASO Analysis

Pros

• Sensitive method to detect known mutations

• Panels of ASO probes useful to detect common mutations

Cons

• Each ASO probe detects only one specific sequence

• Most useful for small sequence changes

Principle of Microarray (Chip) Assay

Synthetic DNA probesSynthetic DNA probes

PrehybridizationPrehybridization PosthybridizationPosthybridization

Probes with Probes with hybridized DNAhybridized DNA

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

R EMOV E FERTIL IZED OO CYTES FR OM OVU LATIN G M OU SE IM ME DIATELY

A FTER FERTIL IZATION

R EMOV E B LA STOC YSTS FROM PREGN A NT M OU SE FOUR DAYS A FTER OVU LATION

FEMA LE PR ON UC LEUSH OLDIN G PIPETTE

INJEC TION N EED LE IMPA LIN G M A LE PR ONU C LEU S OF OOC YTE A ND IN JECTING DN A

OOC YTE

R EIM PLAN T SEVER A L OOC YTE S IN FOS TER M OTHE R

R EIM PLAN T SEVER A L BLA STOC YSTS IN FOS TER M OTHE R

B IRTH

B IRTH

B IRTH

A

C

B

D

SOU TH ER N B LOT OF TAIL D NA N ORTH ERN B LOT

B REED ING

A B C D

C

A

C

B

D

A B C D

SOU TH ER N B LOT OF TAIL D NAA B C D

C ULTU R ED ES CELLS W ITH TAR GETED GEN E

A LTER ATION

INJEC T ES C ELLS INTO B LA STOC YST

+

N ORM AL GEN E

ALTER ED GEN E

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 by

direct sequencing or other techniques that are selected based on whether the mutation is known or unknown.

• Introduction to gene transfer provides a framework for learning about gene therapy and methods for recombinant drug development.

recombinant dna technology stephen b. gruber, md, phd division of molecular medicine and genetics...

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