Download 25. Genetic Testing

Biology 212 General Genetics
Spring 2007
Lecture 25: Genetic Testing
Reading: Chap. 4 pp. 140-143; Chap. 10 p. 384
Other sources: Koblinksky, L. et al. (2005) DNA Forensic and Legal Applications. John
Wiley, Hoboken, NJ.
Lecture Outline:
1. Indirect DNA tests
a. RFLPs
b. Simple sequence repeats and DNA fingerprinting
c. Single nucleotide polymorphisms (SNPs)
2. Direct DNA tests
3. Forensic DNA fingerprinting
Lecture:
There are two major types of DNA testing that are done, indirect tests that look at
a linked marker or polymorphism, and direct tests that look for particular mutations in a
gene.
1. Indirect DNA tests
a. RFLPs = Restriction Fragment Length Polymorphism
Differences in DNA fragment length due to presence/absence of restriction sites
Used to map disease genes and identify physical region of gene
Used in diagnosis of genetic diseases especially when the DNA sequence of a gene was
not yet known.
Example of RFLP Fig. 4.19; Fig. 4.20A
b. Simple sequence repeats = SSRs
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often refered to VNTRs = variable number of tandem repeats
individuals vary with respect to the copy number of a short DNA sequence in a
tandem repeat
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example:
5’ TGTGTGTGTG.....3’
3’ ACACACACAC....5’
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dinucleotide repeat of TG or AC
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occurs in variable numbers of copies
~80,000 copies in the human genome
copies are scattered around about every 500 kb or 500,000 bp
relationship between physical distance in human genome and genetic distance
1% recombination frequency =1 cM ~ 1 million base pairs
Example: Fig. 4.20B
See Fig. 4.21 for use of multiple SSR DNA markers to track alleles in pedigree.
c. Single nucleotide polymorphisms (SNPs)
Variation in a single base pair between two individuals. Can be located anywhere in the
genome. Variation must occur at a frequency of 5% or greater in human populations.
Some lead to gain or loss of a restriction site (RFLPs).
Mapping of all SNPs an important goal of the human genome project because they are
the most common form of genetic differences among people and because they are
distributed almost uniformly along the chromosomes.
2. Direct DNA tests

Used to screen for particular disease alleles
Example: RFLP within beta-globin gene diagnostic of sickle cell anemia
beta-globin gene
exon 1
0.2 kb
exon 2
1.1 kb
intron
Dde I
Dde I
(missing in HbS)
Dde I
DNA test for sickle cell anemia
1. digest normal DNA with Dde I
2. digest DNA from sickle cell patient with Dde I
3. carry out agarose gel electrophoresis and Southern blot
Results:
Normal DNA bands
1.1 kb and 0.2 kb
Sickle cell DNA bands
1.3 kb
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Heterozygote
1.3 kb, 1.1 kb and 0.2 kb
This is a direct test because Dde I cleaves DNA at the site of the mutation.
Dde I recognizes and cleaves at the sequence C/TNAG
Allele
HbA
HbS
Amino acid sequence
pro-glu-glu
pro-val-glu
DNA sequence
CCTGAGGAG
CCTGTGGAG
Other useful tests can also directly test for DNA mutations
 DNA sequencing
 Dot hybridization with allele specific oligonucleotides (ASOs)
 Oligonucleotide microarrays
Direct DNA tests for larger insertions/deletions typically use PCR:
DNA test for Huntington’s disease: Use PCR primers that flank the region contain the
CAG repeats and carry out polymerase chain reaction.
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In normal individual get PCR product that reflects CAG repeats of 10-30 nts.
In HD individuals, get larger PCR product due to expansion in the number of
copies of the CAG sequence (36-121).
From the DNA test, can determine the size of the expansion.
In HD increased #copies of the CAG repeat correlates with earlier age of onset of
the disease symptoms.
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3. DNA tests used in forensics
A. Typically a battery of about 13 DNA tests are run as a panel for DNA fingerprinting.
B. Thirteen loci (genes and markers) and a DNA marker to determine the sex of the
individual (amelogenin) are usually performed. These are the “CODIS” loci (Combined
DNA index system).
C. Two commercial DNA kits provide all the reagents to perform the tests. The tests are
“multiplexed”= multiple PCR reactions for different regions are run simultaneously.
D. Depending on the combination of tests used, a probability of about 1/106 (using
smaller kit with only 6 markers) to 1/1017 identity (both kits with 13 markers) can be
achieved.
Nomenclature of DNA markers: D=DNA; first #=chromosome assignment, S=DNA
sequence tag, second #=identified in order found.
Results for an individual for three marker loci. Norma is homozygous (15, 15) for
D3S1358, and has the 14,16 alleles for vWA (a gene region) and the 25,25 alleles for
FGA (a gene region).
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The next table illustrates some of the CODIS results used to resolve a criminal case also
involving paternity.
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