Molecular Biotechnology and Genomics Lecture 17: DNA Diagnostic Systems and STRs - Prof. J, Study notes of Bioinformatics

Download Molecular Biotechnology and Genomics Lecture 17: DNA Diagnostic Systems and STRs - Prof. J and more Study notes Bioinformatics in PDF only on Docsity! BINF 6010/8010 Fall 2008 Molecular Biotechnology and Genomics Lecture 17: Oct 23rd, 2008 Lecturer: Dr. Weller C W bourse e pages: http://webpages.uncc.edu/~jweller2 Fall 2008 Dr. Weller BINF 6010 ITSC 8010 UNCC 1 Agenda • DNA Diagnostic systems – Forensics/Identification • Microsatellites • RAPD • AFLP • Molecular diagnosis of genetic disease Fall 2008 Dr. Weller BINF 6010 ITSC 8010 UNCC 2 Complex STRs • D21S11: Variable repeat sequences – GenBank (TCTA)4 (TCTG)6 (TCTA)3TA(TCTA)3TCA(TCTA)2------------(TCTA)8---------------TC – ODD (TCTA)4-6 (TCTG)5-6 (TCTA)3TA(TCTA)3TCA(TCTA)2TCCATA(TCTA)8---------------TC – EVEN (TCTA)4-6 (TCTG)5-6 (TCTA)3TA(TCTA)3TCA(TCTA)2TCCATA(TCTA)8TA .TCTA .TC Fall 2008 5Dr. Weller BINF 6010 ITSC 8010 UNCC Identification in Humans • DNA forensic applications use tetra-nt repeats – The PCR reaction is more reliable (no slippage) – The size separation of alleles makes the calls more accurate • This assumes you pick the size of the amplicon well Some tri nucleotides are associated with diseases– - • Forensics tries to avoid personal data not needed for identity • How many do you need? - the level of polymorphism of each STR in the target population(s) – Most variation is in the number of repeat elements Fall 2008 Dr. Weller BINF 6010 ITSC 8010 UNCC 6 Developing the markers • Rules for marker development – Find unique sequence that flanks the repeat elements – The unique sequence must be conserved in the target population H th h t t ibl li l th– ave e s or es poss e amp con eng • So the difference provided by the repeats gives good resolution of alleles – If multiplex markers are to be used then the above rule may be modified so that sets of alleles don’t lover ap. Fall 2008 7Dr. Weller BINF 6010 ITSC 8010 UNCC Allele: 5 6 7 ....…Polymerase Chain Reaction….… Fall 2008 10Dr. Weller BINF 6010 ITSC 8010 UNCC THO1 – A simple locus • ( AATG ) 5-11 Allele: 5 169 bp* 6 173 bp 7 177 bp 8 181 bp 9 185 bp 10 189 bp 11 193 bp *AmpFlSTR Fall 2008 11Dr. Weller BINF 6010 ITSC 8010 UNCC Gel images of microsatellite data www.zoo.cam.ac.uk/ioz/research.htm pleurogene.ca/contents/techniques/genotyping.phpFall 2008 12Dr. Weller BINF 6010 ITSC 8010 UNCC Profiler Plus ID Specimen D3S1358 vWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 Q1 16,16 15,17 21,22 12,12 29,30 15,20 8,12 12,12 8,11 From the first kit we get 9 alleles and from the second 6 alleles, but two are the same, so a total of 13 independent loci. COfiler Specimen D3S1358 D16S539 THO1 TPOX CSF1PO D7S820 Q1 16,16 10,12 8,9.3 9,10 12,12 8,11 Fall 2008 15Dr. Weller BINF 6010 ITSC 8010 UNCC Frequency compendium = D bata ase • The set of alleles can be compared to what has been b d i th i t i f b fo serve n o er exper men s us ng one o a num er o population databases. Fall 2008 16Dr. Weller BINF 6010 ITSC 8010 UNCC D3S1338 African Southwest Southeast American Caucasian Hispanic Hispanic Allele (N=210) (N=203) (N=209%) (N=191) <12 0.0119 0.0123 0.0120 0.0131 12 0.0119 0.0123 0.0120 0.0131 14 0.1214 0.1404 0.0790 0.0838 15 0.2905 0.2463 0.4258 0.3534 15.2 0.0119 0.0123 0.0120 0.0131 16 0.3071 0.2315 0.2656 0.2461 17 0.2000 0.2118 0.1268 0.1623 18 0.0548 0.1626 0.0837 0.1387 19 0.0119 0.0123 0.0144 0.0131 >19 0.0119 0.0123 0.0120 0.0131 Fall 2008 Dr. Weller BINF 6010 ITSC 8010 UNCC 17 Heterozygous Loci • For locus vWA the sample had alleles 15, 17 • From the database, the frequency of allele 15 in the selected population = 0.2361 • From the database, the frequency of allele17 in the sample population = 0.1833 • The genotype frequency is then 0.2362 x 0.1833 x 2 = 0.0866 – Since for a heterozygous allele we have Frequency 1 x Frequency 2 x 2 = Genotype frequency Fall 2008 20Dr. Weller BINF 6010 ITSC 8010 UNCC Hardy-Weinberg reminder Both allele and genotype frequencies in a l ti i t t ipopu a on rema n cons an , or are n equilibrium from generation to generation, unless specific disturbing influences are introduced . p2 + 2pq + q2 = (p + q)2 = 1 Data is not available for all possible genotype combinations, so we use allele frequency instead of genotype frequencies as the basis of likelihood comparisons. θ is used as a measure of the effects of population subdivision: [p2 + [p (1 – p)] θ] is applied at any homozygous locus. Fall 2008 21Dr. Weller BINF 6010 ITSC 8010 UNCC Profile Frequencies • If the loci are independent then the , frequency of the complete profile is the product of the individual frequencies . • 0.0943 x 0.0866 = 0.00817 D3S1358 vWA • Or, 1 in 122 individuals in this population is likely to have this combination of alleles. Fall 2008 22Dr. Weller BINF 6010 ITSC 8010 UNCC Online Resources Fall 2008 25Dr. Weller BINF 6010 ITSC 8010 UNCC Population Survey The following table presents information on 365 population studies from around the world. The studies in this table are grouped by the kits used and by either population location, country of racial origin, or racial group. A reference number followed by, in parenthesis, an x and a number indicates the number of studies of this type in the reference. For example, Morocco 31(x3), indicates three Morocco studies are included in reference 31. Reference numbers for STR population studies involving various commercial STR kits North — | African- 2,15,21,46 1,2(x6), | 23,64 | 4(x2) America | American 4(x2),3, 14,56, 184 El Salvador | 77 Hispanic 2,46 1,2(x5), 23,64 A(x2) 4(x2),5, 184 Bahamas 1 Canada 2 36(x2) Costa Rica 137 Jamaica 1 Mexico 123, 210 2,180 190, 200(x3) Native 2(x4) 2(53).4, 23 4 American 56(x3), 60(x3) Trinidad 1 US. 2(x2),21, 25, 46 1,2(x5), | 23,64 ‘| 4(x2) Caucasian 4(x2),5,14, Fall 2008 Dr-Weller BINF-601047SC-8010 26  203 8 BEI BEE Taiwan Polymorphic Marker Database J bout TPMD | Our aims to construct TPMD are to share useful genotyping information including data of genotyped microsatellite markers, genotyping resources and laboratory supports for promoting genotyping and gene cloning of prevalent diseases. TPMD contains microsatellite markers with di-, tri-, and tetranucleotide repeats deposited by four qualified genotyping laboratories and presented with user-friendly web interfaces for researchers. TThe newly developed TPMD graphic displays with integration of markers into genetic, cytogenetic and physical maps not only show the comparison of marker's heterozygosities in Taiwanese, Japanese, and Caucasian but also facilitate the selection and download of commonly used microsatelite markers for refined mapping and positional candidate cloning of disease genes. To cite TPMD Nucleic Acids Research, 2005, Vol. 33, Database issue D174-D177. Text Display TPMD contains the detail and original allele information of commonly used microsatellite markers. more... Map Display The marker data in TPMD could also be searched and displayed according to cytogenetic, genetic and physical maps. more. Download Download TPMD marker = database. more. Provide protocols, standard makers and useful links from Dr. Yuh-Shan Jou's lab. Ea it mentions why ATGC was established, and explains the purpose, scope of research, and expected achievements of ATGC. SOS LARA Ji Fara Polyrnenphis Marker Database CPO comp Cnn = ew CIDA ~C = ( o 3 i = 3 : = = SE 5 1314 15161718 19202122 x ‘Search Query Text Search and Display Interface # Please key in probe or locus name (ex. D5S2501, ATA84D02, AFMO95tc5) in "Search Query’ than click "go" to show the result. Or click in "chromosome" to list all markers on this chromosome. Ifyou have any comment and question about TPMD. Fall 2008 Contact us [=] @ Contact us [=] Last modified on Feb. 05 2005 Dr. Weller BINF 6010 ITSC 8010 Ifyou have any comment and question about TPMD. ‘National Health Research Institutes. All rights reserved. 27  Tee a Aw” w& ae ie ‘ Microsatellite Resources| News About The Microsatellite Contact Database Microsatellites Consortium Select genome Homo sapiens - Human (NCBI 35) (n.b. more genomes will be added soon) Select location in genome © chromosome © entire genome Select microsatellite features repeat unit size:between | 2 bp (dinucleotide) and|6 bp (hexanucleotide) * no. of repeats:min [10 max [300 repeat unit motif(s): .2. AA,GTA (or leave blank for all) find microsatellites, - Ts all regions x Reset Submit download entire VNTR database (Homo sapiens) Fall 2008 Dr. Weller BINF 6010 ITSC 8010 UNCC 30 RAPD method • Randomly Amplified Polymorphic DNA, one of the arbitrarily primed PCR methods – No prior sequence-specific information apply directly to any genome. – An arbitrary, short (usually a 10-mer, up to 20-mers) primer is used in a PCR reaction on gDNA. – A number of discrete products will result • each comes from a region where there are two segments complementary to the primer (on opposite strands) • close enough for effective amplification to occur under the reaction conditions chosen. Small amounts of input DNA are needed (10ng) a thermocycler and a– , separation/detection system. – Polymorphisms based presence/absence of primer binding sites are identified • You cannot know the number of alleles or whether a single locus is being profiled without further work, so this is usually used in screening diversity, not in individual identification. Fall 2008 31Dr. Weller BINF 6010 ITSC 8010 UNCC RAPD Markers • Dominant markers – Change in a primer binding site means no band – Changes to sequence within the fragment are invisible – Length changes require follow-up to identify as related alleles Fall 2008 32Dr. Weller BINF 6010 ITSC 8010 UNCC Amplified Fragment Length P l hio ymorp sm • This combines the sequence specificity of restriction enzyme sequence recognition with the signal amplification of PCR • The procedure starts with DNA purification, and then uses – DNA digestion with two restriction enzymes (the method can be tuned at this point by the selection of the enzymes) – Adaptors are ligated to the sticky ends that have been generated – these are ~22 bases long and provide known PCR amplification sites – PCR is performed: only fragments of a length to be amplified under the chosen conditions will be seen. • with primers complementary to adaptors but with extensions into the unknown sequence allows tuning of the number of fragments also.l – Fragments are separated on a gel and visualized (Keygene, protocol) Fall 2008 35 AFLP Progeny 9 ————— =| * | | 2B D 233 A aed 28) [apt paced cos 0) gi N Ta) oleae. one oe ahaa lion iy wip es | 2g) eee 11 1[2 D arr es] pete ifica Niilih gdl Ral waka Ad A A h A AM h A M Fo 200bp 300bp 400 bp pote 350 bp r ; AK, mi NSS, ———— x Ananah wonal, RO cA thadhy stbal dal tl® alte S-~saaee— ho a daw A wiht x SSS Wo aT et all DS 250bp 300bp 200bp 145 http:/Awww.fao. org/docrep/007/ae21 6e/ae216e23.jpg .ca/contents/techniques/genotyping.ph, 36 GeneMapper software from ABI dan GeneMarker software from SoftGenetics Fall 2008 37 Molecular Diagnosis of Genetic Disease • Generally there is a locus, or gene, whose variants are known, for single-gene diseases – Sickle-cell disease – Huntingtons disease • For complex diseases there may be several implicated regions, in linkage disequilibrium, with the phenotype, rather than specific sequence variants. Fall 2008 40Dr. Weller BINF 6010 ITSC 8010 UNCC Sequence per se • Fish out a desired region from a sample, and sequence it – Use an oligonucleotide on a solid support that captures a neighboring conserved sequence . – You might clone this if unique sequence needed for restriction or PCR primer recognition is not available (f f for example i a lot o repetitive sequence is in the region) • Use the plasmid sequencing primer sites to obtain the sequence information • To sequence both alleles, if the platform available does not let you see more than one. Fall 2008 41Dr. Weller BINF 6010 ITSC 8010 UNCC Desert Hedgehog Mutation •This is a mis-sense mutation of ATG ACG at the initial Met of the first exon of the DHH gene (signaling molecules regulating morphogenesis) •Reference: F Umehara, et al. AmJHumGenet. Nov 2000 Fall 2008 42 CAPS Markers • Advantages – Most CAPS markers are co-dominant and locus-specific. – Most CAPS genotypes are easily scored and interpreted. – CAPS markers are robust (different labs can get the same results). – The CAPS assay is automatable. • Developing CAPS markers – Sequence the cloned DNA containing the gene. – Design primers and amplify 800–2,000-bp DNA fragments. • Targeting introns or 3' untranslated regions should increase the chance of finding polymorphisms S th PCR d t f t t d t d t i hi h– equence e pro uc rom arge e geno ypes, e erm ne w c REs to use . • Separately digest the amplicons with one or more restriction emzymes. • Screen the digested amplicons for polymorphism – You can use gels stained with ethidium bromide. Fall 2008 45Dr. Weller BINF 6010 ITSC 8010 UNCC Molecular diagnostics: genetic disease • CAPS used for a specific example: • In Sickle-cell anemia the GLU Val mutation removes a Cvn I RE site (CCTNAGG). After amplification and restriction a different banding patter appears. HbA HbS -5Pro-Glu-Glu- vs -5Pro-Val-Glu- -CCTGAGGAG- -CCTGTGGAG- 256 201 181(382) 88 Sl AA AS SS 382 CvnI sites 256 201 181 Fall 2008 46Dr. Weller BINF 6010 ITSC 8010 UNCC 88 Single base-sensitive assay • What if the important difference does not give rise to a change in a RE site? • What if the change is too small to be determined with a hybridization-based assay? – The oligonucleotide ligation assay Fall 2008 47Dr. Weller BINF 6010 ITSC 8010 UNCC ARMS-PCR details • Use two pairs of primers – One pair (pink, red) gives G allele amplicon, the blue/indigo pair gives the A allele – There is a mismatch between the 3-terminal base of an innter primer and the template – To enhance allele specificity a second deliberate mismatch (*) a position -2 from the 3’ end is incorporated in the inner primers – Primers are 26 nt long (or more) so the overall stability is not too affected by the mismatches – it is the extension rate that is manipulated here Th t t i t t diff t di t f th– e wo ou er pr mers are pu a eren s ances rom e polymorphic base, so the amplicon for each allele is a different length. – Use touch-down PCR to enhance the specificity Fall 2008 Dr. Weller BINF 6010 ITSC 8010 UNCC 50 The RETINA Assay 51From Hosono et al, Human Mutation Methods 0, 1-8 (2007) Fall 2008 Dr. Weller BINF 6010 ITSC 8010 UNCC 52