Transcript Nucleic Acids Lectures

Diagnosis of Down syndrome by FISH
Chromosome
21 probe
Chromosome
13 probe
Hybridization
Nucleic acid
Basics
PCR
Electrophoresis
DNA-Protein
interactions
Chromatin
Gene expression
Diagnostic
tools
PCR
DNA polymerase can extend a primer
3’
5’
3’
5’ Single stranded DNA
Primer (~20 nucleotides long)
DNA polymerase
dATP, dGTP, dCTP, dTTP
3’
5’
5’
3’
PCR
DNA polymerase MUST have a primer
3’
5’ Single stranded DNA
DNA polymerase
dATP, dGTP, dCTP, dTTP
3’
5’
No primer, no reaction
PCR
+ DNA polymerase +
dATP, dGTP, dCTP, dTTP
heat
3’
5’
long)
5’
3’
3’
5’
5’ Denatured DNA strand
Primers (~20 nucleotides
3’ Denatured DNA strand
cool
Heat and cool 20 times
Convince yourself
that this will be
nearly the sole
product
DNA-based Tools
• PCR
– Excellent for amplification.
– Good for detecting mutations that result in
small changes in allele size.
– There is a limit to the size of the PCR product
(about 500 bp)
– Semiquantitative (can not distinguish one copy
of a gene from two copies of the gene).
Hybridization
Nucleic acid
Basics
PCR
Electrophoresis
DNA-Protein
interactions
Chromatin
Gene expression
Diagnostic
tools
DNA-based Tools
AMPLIFICATION BY PCR
• Detection of Infectious agents
– Commonly used for detection of clamydia and
gonorrhea
• Obtain sample; prepare DNA
• PCR amplify with primers specific for DNA of
organism
• Measure the amount of DNA produced by the PCR
reactions
DNA-based Tools
Mutation detection by PCR
• Detection of small deletions or insertions
– Example: detection of the deltaF508 cystic
fibrosis conductance regulator (CFTR) allele
Person's genotype:
(N: normal allele; A. affected allele)
N/N
N/A
A/A
(normal) (carrier) (affected)
Control (DNA from
an individual known
to be homozygous
normal
Direction of
electrophoresis
Normal allele
ΔF508 allele
(3bp small than normal)
+
Detection of the ΔF508 allele by PCR. A DNA sample from each person is amplified by PCR using
primers that flank the region that contains the ΔF508 mutation. The PCR products are resolved by
electrophoresis, and visualized by staining the DNA. The homozygous normal individual shows one
band, the same size as a normal control; the heterozygous individual shows two bands, one the size of
the normal control and one three base pairs shorter; the homozygous affected individual shows one band
three base pairs shorter than the normal control.
Hybridization
Nucleic acid
Basics
PCR
Electrophoresis
DNA-Protein
interactions
Chromatin
Gene expression
Diagnostic
tools
DNA-based Tools
STRs (Short Tamdem Repeats)
(Also called SSR (simple sequence repeat) or
microsatellite)
– Paternity testing
– Forensics
– Detection of microsatellite instability
Cells
(e.g. white blood cells)
Isolate DNA
5'
3'
5'
3'
AATGAATGAATG
TTACTTACTTAC
Maternal chromosome containing an STR with
three repeats.
3'
5'
3'
5'
AATGAATGAATGAATG
TTACTTACTTACTTAC
Paternal chromosome containing an STR with
four repeats.
Design PCR primers that will flank the STR to be examined
5'
AATGAATGAATG
3'
3'
TTACTTACTTAC
5'
PCR primers hybridize to the two strands of
the maternal and paternal chromosomes (only
the maternal chromosome is shown).
Many rounds of PCR amplification
AATGAATGAATG
TTACTTACTTAC
PCR products from maternal chromosome
AATGAATGAATGAATG
TTACTTACTTACTTAC
PCR products from paternal chromosome
Electrophoresi
s
PCR product from the paternal
chromosome
PCR product from the maternal
chromosome
How to determine short tandem repeat (STR) lengths. DNA is isolated from an individual, the STR of interest is
amplified by PCR using primers that flank the STR repeat. The sizes of each of the two copies of the STR (one
inherited from the mother, and one inherited from the father) are determined by electrophoresis.
DNA-based Tools
STRs (Short Tandem Repeats)
• On September 15, 1990, near the city of Prague,
the body of a woman was discovered in a trench.
• The identity of the victim was soon established.
• The cause of death was determined to be the
result of ligature strangulation.
DNA-based Tools
STRs (Short Tandem Repeats)
• Between January 1991 and April 1992 the bodies
of seven prostitutes were discovered in wooded
areas in different parts of Austria.
• In June and July 1991, the bodies of three
prostitutes were discovered in Los Angeles.
DNA-based Tools
STRs (Short Tandem Repeats)
• In May 1991, a potential suspect for the
murders in Austria was identified.
• Suspicious looking hair was found in the
suspect’s car.
DNA-based Tools
STRs (Short Tandem Repeats)
DNA-based Tools
STRs (Short Tandem Repeats)
• Two nanograms of DNA was obtained from
hair found in the suspect’s car.
• PCR was used to examine several STR loci.
C
TP
Th
Victim’s DNA
Hair DNA
Allele frequency at TPOX locus
Allele
U.S.
Caucasian
U.S.
Afroamericans
6
7
8
9
10
11
12
13
.0000
.0009
.5285
.1023
.0498
.2802
.0374
.0009
.0603
.0232
.3647
.1888
.0975
.2250
.0405
.0000
TPOX allele 11
.28 X .28 = .08
CSF1PO allele 9: .02
Allele 10: .223
.02 X .223 X 2 = .009
Australian
Aboriginies
(Adelaide)
.0000
.0000
.2490
.5520
.0600
.2390
.0000
.0000
THO1 allele 9.3: .345
.08 X .12 X .009 = 8 X 10-5
.345 X .345 = .12
DNA-based Tools
STRs (Short Tandem Repeats)
• The suspect was convicted and is currently
serving a life sentence in Austria.
Hybridization
Nucleic acid
Basics
PCR
Electrophoresis
DNA-Protein
interactions
Chromatin
Gene expression
Diagnostic
tools
DNA-based Tools
• ASO (Allele Specific Oligonucleotides)
– Used to detect specific alleles.
– Can determine if a person is homozygous or
heterozygous for a particular allele
Blood sample from a cystic fibrosis carrier
Isolate DNA
Normal allele
Variant allele
C
G
+
C
G
A
T
C
PCR with primers that flank the
region to be examined
A
T
+
Genomic DNA
PCR-amplified DNA
(split the sample,
isolate the PCR products by electtrophoresis
and blot.)
Hybridize with ASO that is
complementary to normal allele
C
C
G
T
A
A
T
G
C
Hybridization
Hybridize with ASO that is
complementary to variant allele
No hybridization
A
No hybridization
hybridization
Person's genotype:
(N: normal allele; A. affected allele)
N/N
N/A
A/A
(normal) (carrier) (affected)
N/N
N/A
A/A
(normal) (carrier) (affected)
PCR product
Hybridization with normal ASO
Hybridization with variant ASO
Figure 3.7b. ASO-based detection of variant alleles: results.. Blood samples from three individuals
analyzed by ASO hybridization as described in figure 3.7a. The homozygous normal individual shows
hybridization only with the normal ASO, the heterozygous individual shows hybridization with both
ASOs and the individual who is homozygous affected shows hybridization with only the variant ASO.
DNA-based Tools
• ASO (Allele Specific Oligonucleotides)
– Good Knowledge of the allele to be tested is
required for distinguishing one allele from
another
– Usually used in conjunction with PCR,
electrophoresis and DNA blotting