Single Nucleotide Polymorphisms (SNPs)
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1 Single Nucleotide Polymorphisms (SNPs)
2 Additional Markers 13 core STR loci Obtain further information from additional markers: Y STRs Separating male samples Mitochondrial DNA Working with extremely degraded DNA SNPs
3 What is a SNP? Single base pair variation Outside of coding sequence Exist all over the genome Much more common than STRs ATTGCTAGAT TAACTATCTA
4 Compare SNPs vs. STRs STRs Many alleles per marker (>5) Less common 1 per 15,000 bps PCR product size 100 to 400 bps Multiplex > 10 markers per reaction SNPs Two alleles per marker Much more common 1 per 1000 bps PCR product < 100 bps Potentially multiplex 1000s of SNP per chip
5 Advantages to SNPs PCR products can be very small: Markers will work with extremely degraded DNA samples More common in genome May possibly multiplex hundreds or thousands on one chip Sample processing may be completely automated No stutter products
6 Disadvantages to SNPs Less alleles Each marker is less informative Therefore have to genotype many more SNPs to get same level of information about DNA sample Mixture interpretation is more difficult Multiplexes don t actually work yet Currently uses more of the DNA sample than STRs use
7 How many SNPs? Since each SNP is less informative Because only has two alleles Need to genotype more SNPs to equal distinctive DNA profile Computationally: 25 to 45 SNPs equal 13 core STR loci Actual lab work: 50 or more SNPs equal 12 STRs
8 How many SNPs? Actual number of SNPs needed depends on the allele frequencies Why is this true? If one allele is very common then SNP is NOT a good marker everyone will be the same Therefore different SNPs will be useful in different populations Estimate is 50 to 100 SNPs to equal the discrimination of common STR kits today
9 SNP Markers There are thousands of SNP markers known currently The SNP Consortium (TSC) dbsnp International HapMap Project All these groups are identifying: SNP s genotypes Their locations in the genome Allele frequencies in different populations
10 SNP Genotyping Many different methods to genotyping SNPs: Reverse Dot Blots Direct Sequencing HPLC Genotyping TaqMan Assay Fluorescence Polarization (FP) Mass Spec Microchips Pyrosequencing Allele Specific Hybridization SNaPshot
11 Reverse Dot Blots Allele specific probes are bound to blot paper at different places DNA sample is washed over blot DNA will adhere to probes based on complementary base pairing Positive result produces dark spot on blot at specific position Genotype based on position of spots
12 Direct Sequencing Sequence the region of interest Using Sanger dideoxy sequencing method Then simply read the sequence of the region Homo A Hetero A/C Homo C
13 HPLC Genotyping HPLC = liquid chromatography Inject DNA sample into HPLC column HPLC separates components of any sample based on how long it takes to travel through the column Can see three different bands on column: Homozygous one base Heterozygous Homozygous other base
14 TaqMan Assay Uses a probe that binds to center of DNA sequence you are amplifying Probe contains both: Reporter dye Quencher dye stops reporter from fluorescence Whenever two dyes break apart you see fluorescence of reporter dye Probe is allele specific so that it will only break upon copying exact DNA sequence
15 Fluorescence Polarization (FP) Four different ddntps Four different colored dyes Primer extension across SNP region Monitor what color ddntp is added All other sequence in PCR product is identical between samples Difference in color will tell you genotype Mix of colors shows heterozygote
16 Mass Spec Four different ddntps Four different atomic weights Primer extension across SNP region Monitor which ddntp is added By running PCR product through Mass Spec and calculating exact weight of product Sequencing can be done this way as well
17 Microchips Silicon chip has thousands of oligo probes attached to it Exact position of each probe is known Probes are allele specific PCR products are labeled with fluorescent dyes Washed over microchips Product will bind to specific probe based on complimentary base pairing Position of label will show which probe bound
18 Pryosequencing dntps are added one at a time Each dntp incorporated results in the release of pryophosphate + light This is a way to sequence DNA 1. This individual is homo A 2. Hetero A/T would show both peaks 3. Homo T would show T peak but no A peak
19 Allele-Specific Hybridization Again there are allele specific probes Representing all possible genotypes Attached to beads or a microchip Labeled PCR product is washed over beads/chip PCR product will hybridize to whichever probe has the correct sequence Genotype determined by position of label
20 SNaPshot Also known as mini-sequencing 1. Amplify region around SNP 2. Clean up and remove unused reagents: 1. Exonuclease eats up extra primers 2. SAP eats up extra dntps 3. Add extension primers that go right up to SNP itself 4. Labeled ddntps are added Each nucleotide is a different colored dye
21 SNaPshot Assay After primer has extended across SNP Check color of ddntp that was added: Blue peak Homo G Yellow peak Homo C Two peaks Hetero G/C If you do not clean up PCR products before primer extension Dye blobs will interfere with genotyping Huge dark peaks covering many bases
22 SNaPshot Multiplexing Assay can be multiplexed to genotype many SNPs with same sample Add a poly-t tail to each primer 5 T bases in a row Every product will be 5 bases different in size separates the peaks Check size and color when determining genotype
23 SNP genotyping Know basic mechanism of each method Popular methods for Forensics: Pyrosequencing, TaqMan, SNaPshot Requirements of genotyping method to be useful for forensic samples: Require a small amount of DNA Work well with degraded DNA samples Examine multiple markers within same sample multiplex
24 Applications for SNPs The promise of SNPs in Forensics comes from a few specific applications: Estimating Ethnic Background Predicting some physical traits Better recovery of severely degraded DNA sample
25 Estimating Ethnic Origins SNPs have a much lower mutation rate than STRs SNPs 1 mutation per 10 8 generations STRs 1 mutation per 1000 generations Therefore, SNPs are fixed in certain populations By genotyping a fixed SNP can suggest possible ethnic origins May provide information about unknown prep.
26 Estimating Ethnic Origins Individuals with mixed backgrounds may not have expected phenotypic characteristics Need to be interpreted with caution In relation to additional evidence What other ethical problems might exist? Racial profiling Poor science that cannot actually predict racial background with accuracy
27 Phenotypic Predictions Target genes that control: Pigmentation Melatonin Forensic Science Service ID ing SNP associated with mutation in Melanocortin 1 gene that causes red hair Cannot take into account: Environmental changes like what? Aging
28 Genotyping Degraded Samples Primary advantage that SNPs have for forensics is the ability to genotype even a very small PCR product Less than 100 bps This allows accurate genotyping when: DNA is severely degraded PCR Inhibitors are present Currently, SNP genotyping requires more sample than STRs: Need to genotype more markers
29 Info about SNPs SNPs are already in use for Forensics: Mass disaster body ID Paternity testing Genotyping SNPs will continue to become cheaper and easier Needs to become more standardized Like 13 CODIS STR loci Will not replace STRs any time soon Why not? DNA databases are filled with STR profiles
30 Assessment of Future of SNPs European Network of Forensic Science Institutes (ENFSI) US Scientific Working Group of DNA Analysis Methods (SWGDAM) Got together and examined uses of SNPs Conclusions: SNPs are very useful in certain conditions Will not replace STRs for a long time
31 Other Bi-Allelic Markers There are other markers with two alleles that can also be useful under certain conditions Alu Repeats Alu is a human specific 300 base pair repeat Genotype will be human specific INDELs INsertion or DELetion polymorphisms
32 ALU Repeats Repeated DNA is throughout genome Unknown purpose One type of repeat Alu Once Alu is inserted it is a stable event Is not lost or rearranged easily Clear advantage to Alu repeats: They are only present in human genome Can be used to differentiate completely unknown sample into human vs. non-human
33 Human Alu Repeat (~300 bp) AluI Two possible alleles long (+) allele 400 bp short (-) allele 100 bp Figure 8.3, J.M. Butler (2005) Forensic DNA Typing, 2 nd Edition 2005 Elsevier Academic Press
34 INDELs INsertion or DELetion polymorphisms Segment of DNA ranging from: 1 nucleotide Hundreds of nucleotides Two genotypes: Short vs. long Allele frequencies of many INDELs have been estimated in different ethnic populations
35 Any Questions? Read Chapter 9
36 Questions 1. Why are SNPs being considered for use in human identity testing? 2. What are advantages of SNPs? 3. What are disadvantages of SNPs? 4. Will SNPs replace STRs? Why or why not? 5. What are ethical challenges of SNP genotyping predicting either: 1. Race 2. Physical traits
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