Download 24. DNA testing

BIOL 311 Human Genetics
Fall 2006
Lecture: DNA Testing
Reading: Chap. 18 pp. 510-519
Lecture outline:
1. Materials for testing
2. Detecting mutations
a. DNA sequencing
b. mismatches and heteroduplexes
c. SSCP analysis
d. protein truncation test
e. multiplex PCR
3. DNA tests for specific mutations
Lecture:
1. Materials for testing
DNA tests based mainly on PCR
isolate DNA from
 tissue samples
 blood
 mouthwash
 cheek cells (buccal swabs)
 cvs or amniocentesis
 a cell from an 8 cell embryo
 hair
 semen
 tumor specimens
 Guthrie cards: spot of blood for screening newborns for PKU
Can also test RNA by RT-PCR:
Isolate mRNA from tissue use reverse transcriptase to copy into cDNA add PCR
primers, carry out PCR
Can test for proteins that are enzyme by measuring enzyme activity
2. Detecting mutations
for most genetic diseases, many different mutations can lead to same disease
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Table 18-1 compare mutations that cause CF and DMD
CF
DMD
Autosomal recessive
X-linked recessive
Loss of function mutations
Loss of function mutations
Fairly large gene: 250 kb genomic DNA
Giant gene: 2400 kb genomic DNA
27 exons, 6.5 kb mRNA
79 exons, 14 kb mRNA
Almost all mutations are single nt changes, 65% of mutations are deletions of 1 or
although most common is a 3 nt deletion
more exons
5% duplications
30% nonsense or splice site mutations
New mutations are very rare
New mutations are very frequent
Mosaicism not a problem
Mosaicism is common; especially female
cases
Little intragenic recombination
Recombination hotspot (12% between
markers at either end of gene)
a. DNA sequencing can be used to identify a mutation anywhere in gene
due to completion of human genome project, it's becoming cheaper and easier
for some mutations other methods might be preferred due to speed, cost, or need to have
information based on mRNA or protein.
b. some methods detect heteroduplexes
heteroduplex of mismatch between mutant strand and normal strand
can distinguish based on
gel electrophoresis--special gel types
denaturing HPLC (heterodupleses denature differently than normal duplexes)
chemical cleavage of mismatches
c. SSCP analysis (single strand conformation polymorphism
single-stranded DNA folds into unique structures
PCR amplify region around mutation
Separate by electrophoresis
d. Protein truncation test
Coupled transcription-translation reaction
Normal mRNA normal protein
Defective mRNA truncated protein
Fig. 18.5 scan of DMD gene
e. scan for deletions using multiplex PCR
design PCR primers for each intron-exon junction
products produced are different sizes
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"multiplexing"=multiple primer sets in on reaction, visualize by gel electrophoresis
see Fig. 18.6 screen for DMD deletions; note patients 7 & 9 do not show deletions of
these exons.
3. DNA tests for specific mutations
a. Test for presence or absence of a restriction site Fig. 18-8
b. allele specific oligonucleotide hybridization/DNA chips Fig. 18-9
c. ARMS test=allele specific PCR amplification
a multiplex PCR reaction to detect specific point mutations
Table 18-4 Examples of mutations that show a limited range of mutations
Disease
Sickle cell anemia
Achondroplasia (dwarfism)
Common mutation
Single nt substitution in beta-globin
Single nt substitution in fibroblast growth
factor receptor 3 (FGFR3) gene
Unstable triplet repeat expansion
HD
Myotonic dystrophy
Fragile X
Alpha and beta thalessemia
Particular mutations in alpha and beta
globin genes
Two common mutations: 4 bp insertion,
donor splice site
Δ508 deletion in 80% of cases
Tay-Sachs
Cystic fibrosis
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