Recombinant DNA Technology

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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

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Learning Objectives (1)

polymorphisms.

technologies.

Chromosomes, DNA, and Genes

Cell Nucleus _Chromosomes

Gene

Protein

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Genetic Code

A codon is made of 3 base pairs 64 codons total

1 codon (AUG) encodes methionine and starts translation of all proteins

3 codons stop protein translation 61 codons encode 20 amino acids (redundant code) U A A A U G Met G C A Ala

DNA Transcription and

Translation

mRNA Ribosome Growing chain of amino acids Protein Nuclear

membrane Cell membrane

DNA

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5' end Promoter RNA transcription start site 3' end

Gene Structure

Stop site

Intron Exon 2 Intron

Exon 1 Exon 3 Splice sites Exon 2 Exon 1 Exon 3 mRNA

RNA Processing

Translation Protein DNA Primary mRNA Mature mRNA Processing Transcription

Exon Intron Exon Intron Exon

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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 acid

polymorphisms.

• DNA sequence analysis • Transgenic technologies from Textbook: 5.4 1944 DNA is the genetic material 1949 Abnl Hemoglobin in sickle cell anemia

1953 Double helix 1956 Glu 6 Val in sickle hemoglobin 1966 Completion of the genetic code 1970 First restriction enzyme 1972 Recombinant plasmids 1975 Southern blotting 1981 Transgenic mice 1983 Huntington Disease gene mapped 1985 PCR 1986 Positional cloning (CGD, muscular dystrophy, retinoblastoma 1945 1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000 1987 Knockout mice 1989 Positional cloning without deletion (CF) 1990 First NIH-approved gene therapy experiment 1996 Complete yeast genome sequence 1995 1st_complete bacterial genome sequence 2001 Draft human genome sequence

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Preparing DNA for Analysis

Blood sample Centrifuge and extract DNA from

white blood cells

DNA for analysis

SINGLE-STRANDED DNA PROBES FOR GENE A MIXTURE OF SINGLE-STRANDED DNA MOLECULES + B B B A A C C C D D D E E E F F F

ONLY A FORMS A STABLE

DOUBLE-STRANDED COMPLEXES STABLE COMPLEXESA, C, E ALL FORM STRINGENT HYBRIDIZATION REDUCED-STRINGENCY HYBRIDIZATION

A

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Electrophoresis of DNA

Voltage

+

DNA fragments loaded into wells

Path of migration DNA fragments separate by size and charge

_

Electrophoresis Restriction enzyme digestion

Principle of a Southern blot

hybridize labeled probe to fragment of DNA

Add radio-labeled normal DNA

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Polymerase Chain Reaction

(PCR)

Isolate and denature DNA Anneal and extend primers Repeat as necessary Amplified segments Sequence to be amplified

DNA Sequencing

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5' 3' T G T T C T G A C T T C G A C A A SINGLE-STRANDED DNA OF UNKNOWN SEQUENCE RADIOACTIVELY LABELED PRIMER O CH O2 H H H H H H 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 REACTION MIXTURES d d A T P

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

GEL ELECTROPHORESIS AUTORADIOGRAPHY TO DETECT RADIOACTIVE BANDS 3' 5' BASE d d C T P d d T T P d d G T P LARGER FRAGMENTS SMALLER FRAGMENTS C T G A C T T C G ddG ddG PRODUCTS IN ddGTP REACTION P P 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

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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 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”

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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)

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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 protein Nonfunctional or

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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 12 11 10 Textbook: Figure 5.22

SNP (coding sequence)

Normal mRNA 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 Sequence variant mRNA Protein 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

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Disease-Associated Mutations

A mutation is a change in the normal base pair sequence

Commonly used to define DNA sequence changes that alter protein function

Polymorphism

DNA sequence changes that do not alter

protein function (common definition, not technically correct)

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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)

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Silent Sequence Variants

Normal mRNA 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 the amino acid sequence (a type of polymorphism)

Sequence variant

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 Normal mRNA 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

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Nonsense Mutations

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

Nonsense mRNA Protein Normal mRNA 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 A C

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

Frameshift Mutations

Frameshift A U G U G C A A Met A A G Lys G C G Ala C A U U U U G Leu

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

mRNA Protein Normal mRNA 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

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Splice-Site Mutations

Exon 1 Intron Exon 2 Intron Exon 3

Exon 1 Exon 3

Altered 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

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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)

polymorphisms.

– SSCP – DGGE – CSGE – ASO

– Chip technology

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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)

DNA

Gel

Normal Mutated

mutation

• DNA is denatured into single strands

• Single strands fold; shape is altered by mutations • Mobility of mutant and

normal strands differ in gel

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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

DNA

Denaturing gradient gel

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Denaturing Gradient Gel

1 normal homoduplex band 2 heteroduplex bands 1 mutant homoduplex band

BRCA1 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

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Heteroduplex Analysis (CSGE)

Normal band Mutated bands Single-strand DNA Cold Reannealed DNA Amplify and denature DNA

Evaluating Heteroduplex

Analysis

Pros • >90% sensitivity • Rapid, simple assay • Easily automated for high

throughput use

Cons

• Subsequent sequencing needed to characterize mutation

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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 normal DNA

probes

Amplify DNA and hybridize to membranes

Allele Specific Oligonucleotide

(ASO) Hybridization

Add known mutant DNA probes Patients #1 #2 #3 #1 #2 #3

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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 probes

Prehybridization Posthybridization

Probes with hybridized DNA

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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

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polymorphisms.

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REMOVE FERTILIZED OOCYTES FROM OVULATING MOUSE IMMEDIATELY

AFTER FERTILIZATION

REMOVE BLASTOCYSTS FROM PREGNANT MOUSE FOUR DAYS AFTER OVULATION FEMALE PRONUCLEUS HOLDING 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 DNA A 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 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.