Diagnosis of Down syndrome by FISH
Chromosome 13 probe
Chromosome 21 probe
Nucleic acid Basics
Hybridization
Electrophoresis
PCRDiagnostic
tools
DNA-Proteininteractions
Chromatin
Gene expression
3’ 5’ Single stranded DNA 5’ 3’ Primer (~20 nucleotides long)
PCRDNA polymerase can extend a primer
DNA polymerasedATP, dGTP, dCTP, dTTP
3’ 5’ 5’ 3’
3’ 5’
PCRDNA polymerase MUST have a primer
DNA polymerasedATP, dGTP, dCTP, dTTP
No primer, no reaction
3’ 5’ Single stranded DNA
3’ 5’5’ 3’ Denatured DNA strand
3’ 5’ Denatured DNA strand 5’ 3’ Primers (~20 nucleotides long)
PCR+ DNA polymerase +dATP, dGTP, dCTP, dTTP
heat
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).
Nucleic acid Basics
Hybridization
Electrophoresis
PCRDiagnostic
tools
DNA-Proteininteractions
Chromatin
Gene expression
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
-
+
Detection of the ΔF508 allele by PCR. A DNA sample from each person is amplified by PCR usingprimers that flank the region that contains the ΔF508 mutation. The PCR products are resolved byelectrophoresis, and visualized by staining the DNA. The homozygous normal individual shows oneband, the same size as a normal control; the heterozygous individual shows two bands, one the size ofthe normal control and one three base pairs shorter; the homozygous affected individual shows one bandthree base pairs shorter than the normal control.
Normal allele ΔF508 allele(3bp small than normal)
Person's genotype: N/N N/A A/A(N: normal allele; A. affected allele) (normal) (carrier) (affected)
Control (DNA froman individual knownto be homozygousnormal
Direction ofelectrophoresis
Nucleic acid Basics
Hybridization
Electrophoresis
PCRDiagnostic
tools
DNA-Proteininteractions
Chromatin
Gene expression
DNA-based Tools
STRs (Short Tamdem Repeats)
(Also called SSR (simple sequence repeat) or microsatellite)– Paternity testing– Forensics– Detection of microsatellite instability
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.
Isolate DNA
3' 5'
5' 3'
Design PCR primers that will flank the STR to be examined
3'
5'
3'
5'
Electrophoresis
Cells (e.g. white blood cells)
AATGAATGAATG TTACTTACTTAC
Maternal chromosome containing an STR with three repeats.
AATGAATGAATG
TTACTTACTTAC
AATGAATGAATG TTACTTACTTAC
3' 5'
5' 3'
AATGAATGAATGAATG TTACTTACTTACTTAC
Paternal chromosome containing an STR with four repeats.
PCR primers hybridize to the two strands of the maternal and paternal chromosomes (only the maternal chromosome is shown).
Many rounds of PCR amplification
AATGAATGAATGAATG TTACTTACTTACTTAC
PCR products from maternal chromosome PCR products from paternal chromosome
PCR product from the paternal chromosome PCR product from the maternal chromosome
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 ToolsSTRs (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 locusAllele U.S.
CaucasianU.S.Afroamericans
AustralianAboriginies(Adelaide)
6 .0000 .0603 .00007 .0009 .0232 .00008 .5285 .3647 .24909 .1023 .1888 .552010 .0498 .0975 .060011 .2802 .2250 .239012 .0374 .0405 .000013 .0009 .0000 .0000
TPOX allele 11.28 X .28 = .08
CSF1PO allele 9: .02Allele 10: .223.02 X .223 X 2 = .009
THO1 allele 9.3: .345.345 X .345 = .12 .08 X .12 X .009 = 8 X 10-5
DNA-based Tools
STRs (Short Tandem Repeats)
• The suspect was convicted and is currently serving a life sentence in Austria.
Nucleic acid Basics
Hybridization
Electrophoresis
PCRDiagnostic
tools
DNA-Proteininteractions
Chromatin
Gene expression
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
Isolate DNA
PCR with primers that flank theregion to be examined
Hybridize with ASO that iscomplementary to normal allele
Hybridize with ASO that iscomplementary to variant allele
CG T
Hybridization No hybridization
G
AT
A
No hybridization hybridization
C
AT
CG
CG
ATC
+
+
C A
Normal allele Variant allele
Genomic DNA
PCR-amplified DNA
(split the sample,isolate the PCR products by electtrophoresisand blot.)
Blood sample from a cystic fibrosis carrier
PCR product
Person's genotype: N/N N/A A/A(N: normal allele; A. affected allele) (normal) (carrier) (affected)
Hybridization with normal ASO
N/N N/A A/A(normal) (carrier) (affected)
Hybridization with variant ASO
Figure 3.7b. ASO-based detection of variant alleles: results.. Blood samples from three individualsanalyzed by ASO hybridization as described in figure 3.7a. The homozygous normal individual showshybridization only with the normal ASO, the heterozygous individual shows hybridization with bothASOs 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