1. Taqman Technology and Its Application to Epidemiology Yuko You, M.S., Ph.D. EPI 243, May 15 th, 2008

2. Current Techniques for SNP PCR-RFLP PCR-RFLP Taqman Taqman Other technologies Other technologies Sequencing Sequencing Microarray (DNA chips) Microarray (DNA chips) SNPlex SNPlex Illumina SNP panel Illumina SNP panel

3. PCR-RFLP

4. What is PCR? Polymerase Chain Reaction Polymerase Chain Reaction A laboratory technique that can amplify the amount of DNA from a tiny sample to a large amount within just a few hours. A laboratory technique that can amplify the amount of DNA from a tiny sample to a large amount within just a few hours. Applications for basic science, epidemiology, evolution, linkage analysis, forensics, anthropology Applications for basic science, epidemiology, evolution, linkage analysis, forensics, anthropology

5. How PCR is done ? Primers ---->

6. How PCR is done ?

7. Typical PCR Reaction Mix ComponentCONTENTFUNCTION WaterH2OAdjusted PCR reaction to appropriate concentration PCR bufferKCl, Tris and MgCl 2 For their efficiency in supporting the activity of the Taq polymerase. dNTPdATP, dTTP, dCTP, dGTP deoxynucleotides Provide both the energy and nucleosides for the synthesis of DNA DMSOdimethyl sulphoxideImprove amplification efficiency PrimersShort pieces of DNABind to DNA template allowing Taq DNA polymerase enzyme to initiate incorporation of the deoxynucleotides. TaqA heat stable enzyme that adds the deoxynucleotides to the DNA template DNAThe DNA template which will be amplified by the PCR reaction.

8. Typical PCR program 95  C 55  C 72  C 4C4C 10min 1min 7min  x30 cycles Depend on primers design

9. Primers Primers are short, artificial DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are complementary to the beginning or the end of the DNA fragment to be amplified. Primers are short, artificial DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are complementary to the beginning or the end of the DNA fragment to be amplified. They anneal by adhering to the DNA template at these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand. They anneal by adhering to the DNA template at these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand.

10. Example: CAPN10 25741 acgtgctctg cctgccgaag tgaggaggct gggcacggtg cctgggttcc ccctgcccag 25801 gcccagtttg gttctcttca gcgtggagag atgattctgt cccaggagcc gggaggaggg 25861 tgatgattct gtcccaggag ctgggaggag ggtgggcttg tgggaggggc tggctctgtc 25921 tgtggccgta gctgctgctt agaccctgcc agggttcatg aggccaccgt ggcgggaggc 25981 cagcgaggag ccgtgtccca cagctgatgc ctggtgtttt ctcactagag aggctgctct 26041 gccatacgcg ggcgctgcct ggggcctggg tcaagggcca gtcagcagga ggctgccgga 5’-GTTTGGTTCTCTTCAGCGTGGAG-3’5-CATGAACCCTGGCAGGGTCTAAG-3’ Annealing time calculate Annealing time calculate Tm = 4(G + C) + 2(A + T)°C Tm = 4(G + C) + 2(A + T)°C = 66°C = 62°C

11. PCR-RFLP Restriction fragment length polymorphism Restriction fragment length polymorphism The purpose is to detection of point mutations after the genomic sequences are amplified by the PCR The purpose is to detection of point mutations after the genomic sequences are amplified by the PCR A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The recognition sites are usually 4 to 6 base pairs in length A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The recognition sites are usually 4 to 6 base pairs in length Use gel electrophoresis easily identifies the mutations, since mutant allele generate smaller DNA fragments Use gel electrophoresis easily identifies the mutations, since mutant allele generate smaller DNA fragments

12. List of Restriction Enzyme

13. PCR-RFLP How a restriction enzyme work? How a restriction enzyme work? i.e. EcoRI i.e. EcoRI

14. Genotype scoring Sample results of PCR-RFLP (CAPN10) Sample results of PCR-RFLP (CAPN10) 12 22 11 12 22 11 22 12 11 12 11 12 22 12 11 12 11 22 11 1 = major allele 2 = minor allele

15. Taqman SNP assay

16. TaqMan SNP Genotyping Assays provide optimized assays for genotyping single nucleotide polymorphisms (SNPs). The products use the 5´ nuclease assay for amplifying and detecting specific SNP alleles in purified genomic DNA samples.

17. Taqman SNP assay The TaqMan SNP probe contains a reporter dye at the 5´ end of the probe and a quencher dye at the 3´ end of the probe. The TaqMan SNP probe contains a reporter dye at the 5´ end of the probe and a quencher dye at the 3´ end of the probe. During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, which results in increased fluorescence of the reporter. During the reaction, cleavage of the probe separates the reporter dye and the quencher dye, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.

18. Taqman SNP assay A substantial increase in….. Indicates…… VIC dye fluorescence only Homozygosity for Allele 1 FAM dye fluorescence only Homozygosity for Allele 2 Both fluorescence signals Allele 1-Allele 2 Heterozygosity

19. Probe-Based Assay Chemistry

20. Detector Emission Filter Light Source Excitation Filter How is Fluorescence Data Collected?

22. Standard Procedures for Taqman SNP assay

23. Taqman SNP assay The assays require only three components: 1. 1. 1 to 20 ng of purified genomic DNA template 2. 2. SNP Genotyping Assay Mix (specific for each polymorphism) 3. 3. TaqMan Universal PCR Master Mix The assays require only one amplification step and an endpoint reading to obtain results.

24. Default Taqman SNP program 95  C 92  C 60  C 10min 15sec 1min x40 cycles

25. SNP Genotyping Assay Mix Sequence-specific forward and reverse primers to amplify the SNP of interest Sequence-specific forward and reverse primers to amplify the SNP of interest Two Taqman Probes Two Taqman Probes One probe labeled with VIC® dye detects the Allele 1 sequence One probe labeled with FAM™ dye detects the Allele 2 sequence Assay designed specifically customized to fit the default PCR program Assay designed specifically customized to fit the default PCR program

26. Genotype scoring

27. Compare PCR-RFLP and Taqman PCR-RFLP Taqman SNP assay Advantage Inexpensive for small setting studies Inexpensive for small setting studies Flexible for most kind of SNP genotyping Flexible for most kind of SNP genotyping Easy to perform Easy to perform Suitable for high throughput genotyping Suitable for high throughput genotyping Need less information about target sequence Need less information about target sequence Less prone to human error Less prone to human error Faster, more efficient for SNP genotyping Faster, more efficient for SNP genotyping Disadvantage Need information about target sequence Need information about target sequence Not suitable for high throughput genotyping Not suitable for high throughput genotyping Prone to human error Prone to human error Not possible for all SNPs assays Not possible for all SNPs assays Higher expenses for equipment maintenance Higher expenses for equipment maintenance

28. Limitations of Taqman Assay Not applicable for multi-allele SNPs Not applicable for multi-allele SNPs No deletion No deletion No insertion No insertion No Microsatellites No Microsatellites May not work if too many SNPs closed to the targeting site May not work if too many SNPs closed to the targeting site

29. Other Molecular Technologies 1. Direct Sequencing DNA sequencing is the process of determining the nucleotide order of a given DNA fragment DNA sequencing is the process of determining the nucleotide order of a given DNA fragment DNA fragments can be labeled by using a radioactive or fluorescent tag on the primer, in the new DNA strand with a labeled dNTP, or with a labeled ddNTP. DNA fragments can be labeled by using a radioactive or fluorescent tag on the primer, in the new DNA strand with a labeled dNTP, or with a labeled ddNTP.

30. Other Molecular Technologies 1. Direct Sequencing (Continue)

31. Other Molecular Technologies 2. Microarray Chips Microarrays measure gene expression by taking advantage of the process of hybridization. Hybridization allows researchers to test whether two pieces of DNA are complementary. Microarrays measure gene expression by taking advantage of the process of hybridization. Hybridization allows researchers to test whether two pieces of DNA are complementary. Some form of DNA spotted on a chip (probes) Some form of DNA spotted on a chip (probes) Density of spots important Density of spots important One individual sample, many genotypes per assay One individual sample, many genotypes per assay

32. Other Molecular Technologies 3. SBE (Single base extension) Allele specific primers designed Allele specific primers designed polymerase synthezises the primer on the template exact one base before the SNP polymerase synthezises the primer on the template exact one base before the SNP 4. Pyrosequencing short-read DNA sequencing and mutation/SNP analysis short-read DNA sequencing and mutation/SNP analysis 5. SNPlex Taqman Assay based (48-96 SNPs per reaction) Taqman Assay based (48-96 SNPs per reaction) 6. Illumina SNP panel carried out in 384, 768, and 1536 plex formats using Illumina custom SNP panels or standard validated pre-manufactured panels carried out in 384, 768, and 1536 plex formats using Illumina custom SNP panels or standard validated pre-manufactured panels

33. Adaptation of method Many SNPs (>1000) few samples ( 1000) few samples (< 600) Array platform Array platform Many genotypes per array Many genotypes per array Few arrays per day Few arrays per day Few SNPs ( 600) Few SNPs ( 600) Liquid based platform Liquid based platform Multi-well plates provide flexibility Multi-well plates provide flexibility Robotics can increase throughput Robotics can increase throughput Usually many samples/one SNP per plate Usually many samples/one SNP per plate Not true in “multiplex” situations Not true in “multiplex” situations

34. Multiplexing vs. Pooling Multiplex Multiplex Many genotypes from one reaction carried out in one tube Many genotypes from one reaction carried out in one tube Pooling Pooling Reactions carried out separately, analyzed together Reactions carried out separately, analyzed together