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