Recombinant DNA Technology

1
Recombinant DNA Technology

• Stephen B. Gruber, MD, PhD
• Division of Molecular Medicine and Genetics
• November 4, 2002

2
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.

3
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
  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.

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Chromosomes, DNA, and Genes
Gene
Nucleus
Cell
Chromosomes
Protein
Adapted from Understanding Gene Testing, NIH, 1995
5
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)
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DNA Transcription and Translation
Growing chain of amino acids
mRNA
Protein
Ribosome
Nuclear membrane
Cell membrane
DNA
Adapted from Understanding Gene Testing, NIH, 1995
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Gene Structure
RNA transcription start site
Stop site
Splice sites
Promoter
Intron
Exon 2
Intron
Exon 1
Exon 3
5' end
3' end
Exon 2
Exon 1
Exon 3
mRNA
8
RNA Processing
Exon
Intron
Exon
Intron
Exon
DNA
Transcription
Primary mRNA
AG
GU
Processing
Mature mRNA
Translation
Protein
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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

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1983 Huntington Disease gene mapped
2001 Draft human genome sequence
1989 Positional cloning without deletion (CF)
1981 Transgenic mice
1956 Glu 6 Val in sickle hemoglobin
1995 1st complete bacterial genome sequence
1970 First restriction enzyme
1944 DNA is the genetic material
1975 Southern blotting
1985 PCR
1953 Double helix
1995
1945
1960
1970
1985
1990
2000
1950
1955
1965
1975
1980
1996 Complete yeast genome sequence
1972 Recombinant plasmids
1986 Positional cloning (CGD, muscular dystrophy,
retinoblastoma
1990 First NIH-approved gene therapy experiment
1966 Completion of the genetic code
1949 Abnl Hemoglobin in sickle cell anemia
1987 Knockout mice
from Textbook 5.4
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Preparing DNA for Analysis
DNA for analysis
Centrifuge and extract DNA from white blood cells
Blood sample
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Textbook Figure 5.8
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Electrophoresis of DNA
_
DNA fragments loaded into wells
DNA fragments separate by size and charge
Voltage
Path of migration

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Principle of a Southern blothybridize labeled
probe to fragment of DNA
Electrophoresis
Restriction enzyme digestion
Add radio-labeled normal DNA probes
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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
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DNA Sequencing
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Textbook Figure 5.17
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DNA Sequencing
AG
ATC TTA GTG TCC C
ATC TTA GAG TGT CCC
delG
delA
Mutant (185delAG)
Start
Normal
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

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Polymorphisms and Mutations

• Sequence variation-- differences among
  individuals (DNA, amino acid)
• gt 0.01 polymorphism
• lt 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

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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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Textbook Figure 5.19
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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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Disease-Associated Mutations Alter Protein
Function
Functional protein
Nonfunctional or missing protein
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P1
P2
A
(TCTA)
10
(TCTA)
B
11
C
(TCTA)
12
D
(TCTA)
13
E
(TCTA)
14
F
(TCTA)
15
15
14
13
12
11
10
AB
CD
EF
AF
CE
Textbook Figure 5.22
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SNP (coding sequence)
mRNA Protein
Normal
G
C
A
G
Ala
Sequence variant
mRNA Protein
Silent DNA sequence polymorphism
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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
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Polymorphism
DNA 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 gt 1

Common usage
lt 1
gt 1
Rare or private polymorphism
polymorphism
Normal
??
Disease
disease
Factor V R506Q thrombosis, 3 allele frequency
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Mutations

• Normal
• Missense
• Nonsense
• Frameshift (deletion)
• Frameshift (insertion)

• THE BIG RED DOG RAN OUT.
• THE BIG RAD DOG RAN OUT.
• THE BIG RED.
• THE BRE DDO GRA.
• THE BIG RED ZDO GRA.

Point mutation a change in a single base pair
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Silent Sequence Variants
mRNA Protein
Normal
G
C
A
G
Ala
Sequence variant
mRNA Protein
Sequence variant a base pair change that does
not change the amino acid sequence (a type of
polymorphism)
Adapted from Campbell NA (ed). Biology, 2nd ed,
1990
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Missense Mutations
mRNA Protein
Normal
mRNA Protein
Missense
A
G
C
G
A
C
Ser
Ala
Missense changes to a codon for another amino
acid (can be harmful mutation or neutral
polymorphism)
Adapted from Campbell NA (ed). Biology, 2nd ed,
1990
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Nonsense Mutations
mRNA Protein
Normal
mRNA Protein
Nonsense
Nonsense change from an amino acid codon to a
stop codon, producing a shortened protein
Adapted from Campbell NA (ed). Biology, 2nd ed,
1990
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Frameshift Mutations
mRNA Protein
Normal
mRNA Protein
Frameshift
Frameshift insertion or deletion of base pairs,
producing a stop codon downstream and (usually)
shortened protein
Adapted from Campbell NA (ed). Biology, 2nd ed,
1990
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Splice-Site Mutations
Exon 1
Intron
Exon 2
Intron
Exon 3
Exon 2
Exon 3
Exon 1
Altered mRNA
Splice-site mutation a change that results in
altered RNA sequence
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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

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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
  acid polymorphisms.
• Know how DNA sequence analysis is performed and
  be familiar with methods of screening for
  differences.  
• SSCP
• DGGE
• CSGE
• ASO
• Chip technology
• methods for gene transfer and the power of
  transgenics

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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

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Single Strand Conformational Polymorphism (SSCP)
Normal
Mutated

• DNA is denatured into single strands
• Single strands fold shape is altered by
  mutations
• Mobility of mutant and normal strands differ in
  gel

DNA
Gel
mutation
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Evaluating SSCP

• Cons
• Subsequent DNA sequencing needed to characterize
  mutation
• Sensitivity drops with longer DNA sequences

• Pros
• Rapid, simple, and widely available for many
  genes
• Detects 60-95 of mutations in short DNA strands

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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

Normal
Mutated
DNA
Denaturing gradient gel
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Denaturing Gradient Gel
BRCA1 mutation carrier
1 normal homoduplex band 2 heteroduplex bands 1
mutant homoduplex band
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Evaluating DGGE

• Cons
• Not efficient for analyzing large DNA fragments
• Subsequent DNA sequencing needed to characterize
  mutation
• Labor-intensive set-up

• Pros
• Highly sensitive (gt90)
• Better resolution than SSCP

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Heteroduplex Analysis (CSGE)
Amplify and denature DNA
Cold
Single-strand DNA
Reannealed DNA
Mutated bands
Normal band
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Evaluating Heteroduplex Analysis

• Pros
• gt90 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

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Allele Specific Oligonucleotide(ASO)
Hybridization
Patients
1
2
3
Add radio-labeled normal DNA probes
1
2
3
Add known mutant DNA probes
Amplify DNA and hybridize to membranes
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Evaluating ASO Analysis

• Cons
• Each ASO probe detects only one specific sequence
• Most useful for small sequence changes

• Pros
• Sensitive method to detect known mutations
• Panels of ASO probes useful to detect common
  mutations

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Principle of Microarray (Chip) Assay
Prehybridization
Posthybridization
Synthetic DNA probes
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
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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
  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.

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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.