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Download Lecture 8 (2/15/10) "DNA Forensics, Cancer, and Sequencing"
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Announcements: Lec. 8, 02/15/10 Due today: Your article on what you will give your talk and research paper Plus general review article plus textbook/web page(s) ½ pg write-up March 15th : Student Presentations March 17th : Student Presentations We will likely have one evening section—pizza included! Next Wednesday– quiz on Chpt 3 of ECB; next homework due. No homework assigned. Next Monday– NO Class (I’m at Biophysical Society Meeting) Today: Applications of DNA: Forensics, FISH, DNA “chips”; Sequence, dead or alive http://www.cnn.com/video/#/video/world/2010/02/12/rush.greenland.oldest.human.itn?hpt=C2 Forensic DNA Typing Did you kill (rape, father…) that person? How DNA can “definitively” say. Adapted from: National Institutes of Science & Technology Target Region for PCR You can be identified by your own unique various regions http://www.cstl.nist.gov/div831/strbase/intro.htm DNA Use in Forensic, Paternity… Cases • Most Forensic cases are rape cases (>2 out of 3) Looking for match between evidence and suspect -- matching suspect with evidence • Paternity testing -- identifying father • Military DNA “dog-tag.” Challenges •Mixtures must be resolved •DNA is often degraded (stored wet- have mold, nuclease) •Inhibitors to PCR are often present Sources of Biological Evidence • • • • • • • Blood Semen Saliva Urine Hair Teeth (useful in fires). Bone (Yes, there are cells in bone. Decalcify it. 100,000 year old people, Dinosaurs- has DNA!) • Tissue All felony arrests- cheek swab. Short Tandem Repeats (STRs) (say chromo 3) Identical in all people AATG 7 repeats Identical in all people 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length Heterozygote = alleles differ and can be resolved from one another Variation Among STRs Choosing which STRs: Significant statistical variation – but not too many. Freq. that are measured in pop. : Loc 1 -10%. Loc 2 – 10%; locus 1+2 -1/100. Random match with 13 primers (used now): 1/1013. (There are 6 billion people, ~ 6 x 109 people.) Watch out for different racial types! Multiplex PCR • Over 10 Markers Can Be Copied at Once • Sensitivities to levels less than 1 ng of DNA • Ability to Handle Mixtures and Degraded Samples • Different Fluorescent Dyes Used to Distinguish STR Alleles with Overlapping Size Ranges Most rxns: require 2 PCR (tubes) 7 or 8 primer pairs in one tube– need total of about 2 tubes for 13 different STRs. $20-$25 per rxn in lab. $150 incl labor. Cost for forensic up to $1000. 13 CODIS Core STR Loci with Chromosomal Positions TPOX D3S1358 D8S1179 D5S818 FGA CSF1PO TH01 VWA D7S820 AMEL D13S317 D16S539 D18S51 D21S11 AMEL D tells chromosome 21— Can tell Down’s syndrome. (No Down’s here.) Human Identity Testing with Multiplex STRs Amelogenin protein is involved in tooth enamel and happens to be on sex chromosome – top: 2 peaks: x (106 bp) and y (112 bp); Bottom only 1 peak cause they have two X chromosomes. ® AmpFlSTR SGM Plus™ kit Two different individuals DNA Size (base pairs) amelogenin D19 106 bp 112 bp D3 D8 TH01 VWA D21 D16 D18 D2 FGA probability of a random match: ~1 in 3 trillion amelogenin D3 D19 D8 VWA TH01 Results obtained in less than 5 hours with a spot of blood the size of a pinhead D16 D21 FGA D18 Simultaneous Analysis of 10 STRs and Gender ID D2 FBI’s CODIS DNA Database Combined DNA Index System --all 50 states can upload their convicted felony and seq. of unsolved cases…. In Florida to convicted felon. • Used for linking serial crimes and unsolved cases with repeat offenders • Launched October 1998 • Links all 50 states • Requires >4 RFLP markers and/or 13 core STR markers • Current backlog of >600,000 samples Except for police errors, and sufficient racial typing, it’s a done deal Clinical Applications (Mitotic) Chromosomes can be identified by their unique sequences Each chromosome can be labeled uniquely The location of genes on a chromosome can be determined Fluoresence in Situ Hybridization (FISH) Green- # 22 marker- 22q13 Red- DiGeorge Syndrome region (if missing) at 22q11.2 (Person has 2 normal) FISH A C B A). Chromosone 4 “painted”. B) From same person in A, but hybridized with a probe for the terminal part of chromosome 4q. Only one green signal one chromosome 4 is missing material from the terminal end of 4q. C) Xcen chromosome 22 Other: Steroid Sulfatase gene. Two X chromosomes, 1 St.Su. gene female carrier for Steroid Sulfatase Deficiency. http://members.aol.com/chrominfo/metafish.htm Nature Genetics Supplement 21:33-37 (January 1999) Molecular portraits of human breast tumours Gene chips can be used to follow genetic changes during cancer and cancer treatment Different Tumors FIG. 1. Variation in expression of 1,753 genes in 84 experimental samples. Affymetrix, 1992. Marriage of Silicon and Genes. Semiconductor manufacturing techniques could be united with advances in combinatorial chemistry to build vast amounts of biological data on a small glass chip. Different Genes Data are presented in a matrix format: each row represents a single gene, and each column an experimental sample. Green squares, transcript levels below the median; black squares, transcript levels equal to the median; red squares, transcript levels greater than the median; grey squares, technically inadequate or missing data. Brown & Botstein, Nature, 406, 747, 2000 Gene Chips can be used to follow genetic changes during development Sequencing: Illumina (and other sources). Decoding 4,000 year old DNA (From Nature, 2009) Less 10 years after first living person’s DNA sequenced “Inuk” died on an island off Greenland called Qeqertasussuk. He left bits of hair and bone that the permafrost preserved, including his complete genome. Inuk’s genes reveal he was a fairly young man, robustly built to exist in a frigid climate, with A-positive blood, dark skin, brown eyes, and thick, black hair on a scalp genetically susceptible to baldness. He was a palaeoEskimo, and by comparing his genome to other living people, they deduced that he was member of the Arctic Saqqaq, the first known culture to settle in Greenland whose ancestors had trekked from Siberia around the Arctic circle in pursuit of game. Contamination a big problem: The best place to find it is entombed in ice, where it is preserved by the cold and protected from contamination. Hair doesn't as readily absorb contaminants, and its surface can be bleached clean. They also tagged the millions of fragments of extracted DNA with a barcode-like sequence to distinguish them from stray modern human DNA. Class evaluation 1. What was the most interesting thing you learned in class today? 2. What are you confused about? 3. Related to today’s subject, what would you like to know more about? 4. Any helpful comments. Answer, and turn in at the end of class.