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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 1 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 2 "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 3