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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
technologies.
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.
Chromosomes, DNA, and Genes
Gene
Cell
Nucleus
Chromosomes
Protein
Adapted from Understanding Gene Testing, NIH, 1995
Genetic Code
A codon is made of 3 base pairs
64 codons total
1 codon (AUG) encodes
methionine and starts
translation of all proteins
61 codons encode 20
amino acids
(redundant code)
3 codons stop
protein
translation
A U G
G C A
U A A
Met
Ala
DNA Transcription and
Translation
Growing
chain of
amino acids
mRNA
Ribosome
DNA
Nuclear
membrane
Adapted from Understanding Gene Testing, NIH, 1995
Protein
Cell membrane
Gene Structure
RNA transcription
start site
Splice sites
Stop site
Promoter
Exon 1
Intron
Exon 2
Intron
5' end
Exon 3
3' end
Exon 1
Exon 2
mRNA
Exon 3
RNA Processing
Exon
Intron
Exon
Intron
DNA
Transcription
Primary
mRNA
GU
AG
Processing
Mature
mRNA
Translation
Protein
Exon
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
1956
Glu 6 Val in
sickle hemoglobin
1944
DNA is the
genetic material
1945
1953
Double helix
1950
1949
Abnl Hemoglobin
in sickle cell anemia
1955
1960
1965
1966
Completion of the
genetic code
1983
Huntington
Disease gene
mapped
1981
Transgenic mice
1970
First restriction
enzyme
1970
1975
Southern
blotting
1975
1972
Recombinant
plasmids
1980
2001
Draft human
genome sequence
1989
Positional cloning
without deletion (CF)
1995
1st complete
bacterial
genome sequence
1985
PCR
1985
1986
Positional cloning
(CGD, muscular
dystrophy,
retinoblastoma
1990
1995
2000
1996
1990
Complete yeast
First NIHgenome sequence
approved
gene therapy
experiment
1987
Knockout
mice
from Textbook: 5.4
Preparing DNA for Analysis
Blood sample
Centrifuge and
extract DNA from
white blood cells
DNA for analysis
SINGLE-STRANDED
DNA PROBES
FOR GENE A
C
D
+
B
MIXTURE OF
SINGLE-STRANDED
DNA MOLECULES
E
A
F
C
C
D
B
B
D
A
A
E
E
F
ONLY A FORMS A STABLE
DOUBLE-STRANDED COMPLEXES
STRINGENT HYBRIDIZATION
F
A, C, E ALL FORM
STABLE COMPLEXES
REDUCED-STRINGENCY HYBRIDIZATION
Textbook: Figure 5.8
Electrophoresis of DNA
DNA fragments loaded into wells
_
DNA fragments
separate by size
and charge
Voltage
Path of migration
+
Principle of a Southern blot
hybridize labeled probe to fragment of DNA
Electrophoresis
Restriction enzyme
digestion
Add radio-labeled
normal DNA
probes
Polymerase Chain Reaction
(PCR)
Isolate and
denature DNA
Sequence to be
amplified
Anneal and
extend primers
Repeat as
necessary
Amplified
segments
DNA Sequencing
SINGLE-STRANDED DNA
OF UNKNOWN SEQUENCE
5'
C T G A C T T C G A C A A
3'
RADIOACTIVELY LABELED
3' T G T T
PRIMER
DNA POLYMERASE I
dATP
dGTP
dCTP
dTTP
ddATP
ddCTP
ddTTP
P
P
P
O
5'
CH 2 O BASE
H
H
H
H
H
H
DIDEOXYNUCLEOTIDE (ddNTP)
ddGTP
REACTION
MIXTURES
C T G A C T T C G A C A A
LARGER
FRAGMENTS
ddG
ddG
ddG
ddGTP
ddTTP
AUTORADIOGRAPHY
TO DETECT
RADIOACTIVE BANDS
ddCTP
ddATP
GEL
ELECTROPHORESIS
PRODUCTS IN ddGTP REACTION
C
T
G
A
C
T
READ SEQUENCE OF ORIGINAL
SINGLE-STRANDED DNA
(COMPLEMENT OF PRIMERGENERATED SEQUENCE LADDER)
T
SMALLER
FRAGMENTS
C
G
Textbook: Figure 5.17
DNA Sequencing
AG
ATC TTA GTG TCC C
ATC TTA GAG TGT CCC
A
T
C
G
A
T
C
G
delA
Start
Normal
Start Mutant (185delAG)
delG
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”
• 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
– SNPs
Tandem Repeat]))
(Single Nucleotide Polymorphism)
Textbook: Figure 5.19
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
– SNPs
Tandem Repeat]))
(Single Nucleotide Polymorphism)
Disease-Associated Mutations
Alter Protein Function
Functional protein
Nonfunctional or
missing protein
P1
P2
(TCTA)10
A
(TCTA)11
B
(TCTA)12
C
(TCTA)13
D
(TCTA)14
E
(TCTA)15
F
15
14
13
12
11
10
AB
CD
EF
AF
CE
Textbook: Figure 5.22
SNP (coding sequence)
Normal
mRNA
Protein
Sequence
variant
A U G
A A G
U U U
G G C
G C A
U U G
C A A
Met
Lys
Phe
Gly
Ala
Leu
Gln
A U G
A A G
U U U
G G U
G C A
U U G
C A A
Met
Lys
Phe
Gly
Ala
Leu
Gln
mRNA
Protein
Silent DNA sequence polymorphism
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)
Functional protein
Functional protein
Polymorphism
• Variation in population
– phenotype
– genotype (DNA sequence polymorphism)
• Variant allele > 1%
Common usage:
< 1%
“Normal”
Rare or “private”
polymorphism
Disease
disease
> 1%
polymorphism
??
Factor V R506Q: thrombosis,
3% allele frequency
Mutations
Normal
THE BIG RED DOG RAN OUT.
Missense
THE BIG RAD DOG RAN OUT.
Nonsense
THE BIG RED.
Frameshift (deletion)
Frameshift
(insertion)
THE BRE DDO GRA.
THE BIG RED ZDO GRA.
Point mutation: a change in a single base pair
Silent Sequence Variants
Normal
mRNA
Protein
Sequence
variant
A U G
A A G
U U U
G G C
G C A
U U G
C A A
Met
Lys
Phe
Gly
Ala
Leu
Gln
A U G
A A G
U U U
G G U
G C A
U U G
C A A
Met
Lys
Phe
Gly
Ala
Leu
Gln
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
Missense Mutations
Normal
mRNA
Protein
A U G
A A G
U U U
G G C
G C A
U U G
C A A
Met
Lys
Phe
Gly
Ala
Leu
Gln
A U G
A A G
U U U
A G C
G C A
U U G
C A A
Met
Lys
Phe
Ser
Ala
Leu
Gln
mRNA
Missense
Protein
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
Nonsense Mutations
Normal
mRNA
Protein
A U G
A A G
U U U
G G C
G C A
U U G
C A A
Met
Lys
Phe
Gly
Ala
Leu
Gln
A U G
U A G
U U U
G G C
G C A
U U G
C A A
mRNA
Nonsense
Protein
Met
Nonsense: change from an amino acid codon to a stop
codon, producing a shortened protein
Adapted from Campbell NA (ed). Biology, 2nd ed, 1990
Frameshift Mutations
Normal
mRNA
Protein
Frameshift
A U G
A A G
U U U
G G C
G C A
U U G
C A A
Met
Lys
Phe
Gly
Ala
Leu
Gln
A U G
A A G
U U G
G C G
C A U
U G C
Met
Lys
Leu
Ala
mRNA
Protein
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
A A
Splice-Site Mutations
Exon 1
Intron
Exon 2
Intron
Exon 3
Exon 2
Altered mRNA
Exon 1
Exon 3
Splice-site mutation: a change that results in altered RNA sequence
Other Types of Mutations
•
Mutations in regulatory regions of the gene
•
Large deletions or insertions
•
Chromosomal 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
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
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)
Normal
DNA
Mutated
• DNA is denatured into
single strands
• Single strands fold; shape
is altered by mutations
• Mobility of mutant and
normal strands differ in
gel
Gel
mutation
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)
Normal
Mutated
DNA
• 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
Denaturing gradient gel
Denaturing Gradient Gel
BRCA1 mutation carrier
1 normal homoduplex band
2 heteroduplex bands
1 mutant homoduplex band
Evaluating DGGE
Pros
Cons
• Not efficient for
• Highly sensitive (>90%)
analyzing large DNA
• Better resolution than SSCP
fragments
• Subsequent DNA
sequencing needed to
characterize mutation
• Labor-intensive set-up
Heteroduplex Analysis (CSGE)
Amplify and
denature
DNA
Cold
Single-strand DNA
Reannealed DNA
Mutated bands
Normal band
Evaluating Heteroduplex
Analysis
Pros
Cons
• >90% sensitivity
• Subsequent sequencing
needed to characterize
mutation
• Rapid, simple assay
• Easily automated for high
throughput use
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
Allele Specific Oligonucleotide
(ASO) Hybridization
Patients
#1
#2
#3
#1
#2
#3
Add radio-labeled
normal DNA
probes
Amplify DNA and hybridize
to membranes
Add known
mutant DNA
probes
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
Prehybridization
Posthybridization
Synthetic DNA probes
Probes with
hybridized 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
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.
CULTURED ES CELLS
WITH TARGETED GENE
ALTERATION
REMOVE BLASTOCYSTS
FROM PREGNANT MOUSE
FOUR DAYS
AFTER OVULATION
REMOVE FERTILIZED OOCYTES FROM
OVULATING MOUSE IMMEDIATELY
AFTER FERTILIZATION
HOLDING
PIPETTE
FEMALE PRONUCLEUS
INJECTION NEEDLE
IMPALING MALE
PRONUCLEUS OF OOCYTE
AND INJECTING DNA
OOCYTE
REIMPLANT SEVERAL OOCYTES
IN FOSTER MOTHER
INJECT ES CELLS
INTO BLASTOCYST
REIMPLANT SEVERAL BLASTOCYSTS
IN FOSTER MOTHER
BIRTH
BIRTH
B
A
D
C
SOUTHERN BLOT
OF TAIL DNA
A B C D
+
NORTHERN BLOT
A B C D
BIRTH
B
A
BREEDING
D
C
C
SOUTHERN BLOT
OF TAIL DNA
A B C D
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.