Post on 22-Dec-2015
transcript
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
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
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
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
Textbook: Figure 5.17
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
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 Tandem Repeat]))
– SNPs (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 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
Textbook: Figure 5.22
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
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
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
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 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). Adapted from Campbell NA (ed). BiologyBiology, 2nd ed, 1990, 2nd ed, 1990
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
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 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). Adapted from Campbell NA (ed). BiologyBiology, 2nd ed, 1990, 2nd ed, 1990
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
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
Adapted from Campbell NA (ed). Adapted from Campbell NA (ed). BiologyBiology, 2nd ed, 1990, 2nd ed, 1990
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
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)
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
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.
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.