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EPI 243, April 21, 2009 Taqman Technology and Its Application to Epidemiology Yuko You, M.S., Ph.D. Current Techniques for SNP PCR-RFLP Taqman Other technologies Sequencing Microarray (DNA chips) SNPlex Illumina SNP panel DNA polymorphisms DNA polymorphisms More than 99% of nucleotides in DNA are the same in all humans. The DNA loci that vary from person to person are said to be “Polymorhic”, and the alternate sequence of the same locus are called Allele. Technically, the term “Polymorphisms” is generally restricted to those variations that are relatively common (> 1% of individuals) DNA polymorphisms Common variant allele usually are named “Wild-type” or “Normal” allele, while rare or less common variant allele are named “Mutant” or “Variant" allele. Person with two copies of the same allele are “Homozygous” and have different alleles on the two chromosomes are “Heterozygous”. Common types of polymorphisms SNP: Single Nucleotide Polymorphism or point mutation A T Transition (A↔G or C↔T) Transversion (less common) Insertion/Deletion Multiple Alleles C Microsatellite – variable number of tandom repeats G SNP SNPs make up 90% of all human genetic variations SNPs with a minor allele frequency of ≥ 1% occur every 100 to 300 bases along the human genome On average, where two of every three SNPs substitute C with T SNP Results from SNP Synonymous Nonsynomous Missense (change one amino acid to another) Nonsense(changing an amino acid to stop) Frameshift (results from insertion/deletion) SNP Naming Restriction site Location EX5+76_78delACT denotes an ACT deletion from nucleotides 76 to 78 of exon 5 Insertions EX3+76A>C denotes that at nucleotide 76 of exon 3, an A is changed to a C G487A Deletions Rs (dbSNP maps each submitted SNP assay to the genome and assigns a RefSNP accession ID (rs number) to each submitted SNP assay ) Rs1800629=TNF-α -308 Substitutions TNF-α -308 rs number ALDH2-MboII 76_77insT denotes that a T was inserted between nucleotides 76 and 77 Amino acid change Pro187Ser denotes that SNP causes amino acid changes from Proline to Serine at amino acid 187 SNP Naming For example: TNF-α Ex3+19C>T (nucleotide 19 of exon 3, an C is changed to a T) rs4645843 Pro84Leu P84L SNP Naming Allele naming By the nucleotide: AA, AC, CC By amino acid: Pro/Pro, Pro/Ser, Ser/Ser By default: 11, 12, 22 (1 as wildtype, 2 as variant) Or 00, 01, 11 (0 as wildtype, 1 as variant) By Minor allele frequency count: 0, 1, 2 Haplotypes A combination of alleles at multiple linked loci that are transmitted together. Haplotype may refer to as few as two loci or to an entire chromosome depending on the number of recombination events that have occurred between a given set of loci. The group of alleles of linked genes contributed by either parent; the haploid genetic constitution contributed by either parent Haplotype Haplotypes Example for 2 SNPs haplotype combination 3’ 5’ SNP1 SNP2 SNP1 AA SNP2 CC CT TT A C A C A T A C A T A T AG A C A C A T G C G T G T or A G T C GG G C G C G T G C G T G T PCR-RFLP What is PCR? 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. Applications for basic science, epidemiology, evolution, linkage analysis, forensics, anthropology How PCR is done ? Primers ----> How PCR is done ? The elements for PCR-RFLP 1. 2. 3. 4. 5. 6. PCR reaction mix DNA PCR program RFLP Agarose gel electrophoresis Genotype scoring 1. PCR Reaction Mix 1. PCR Reaction Mix Component CONTENT FUNCTION Water H2O Adjusted PCR reaction to appropriate concentration PCR buffer KCl, Tris and MgCl2 For their efficiency in supporting the activity of the Taq polymerase. dNTP dATP, dTTP, dCTP, dGTP deoxynucleotides Provide both the energy and nucleosides for the synthesis of DNA DMSO dimethyl sulphoxide Improve amplification efficiency Primers Short pieces of DNA Bind to DNA template allowing Taq DNA polymerase enzyme to initiate incorporation of the deoxynucleotides. Taq A heat stable enzyme that adds the deoxynucleotides to the DNA template DNA The DNA template which will be amplified by the PCR reaction. 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. 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. Taq Polymerase – “Taq” Taq polymerase is a thermostable DNA polymerase named after the thermopilic bacterium “Thermus aquaticus “ which lives in hot spring. an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR Taq's temperature optimum for activity is 75-80°C, with a half-life of 9 minutes at 97.5°C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72°C 3. PCR program (example) Cycle Temp Time NOTE First denaturing 94C 5 min Many researchers use a 2-5 minutes first denaturing step before the actual cycling starts. This is supposed to help denaturing the target DNA better (especially the hard to denature templates). Denaturing 94C 1 min Annealing 55C 1 min Extension 72C 1 min Last extension 72C 5 min Storage 4C An annealing time of 30-60 seconds was sufficient for all primer pairs tested so far. The annealing temperature can be chosen based on the melting temperature of the primers Supposedly to help finish the elongation of many or most PCR products initiated during the last cycle Typical PCR program x30 cycles 95C 95C 10min 1min 72C 55C 1min 72C 7min 1min 4C Depend on primers design 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’ = 66°C 5-CATGAACCCTGGCAGGGTCTAAG-3’ Annealing time calculate Tm = 4(G + C) + 2(A + T)°C = 62°C Primitive PCR machine 4. PCR-RFLP Restriction fragment length polymorphism 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 Use gel electrophoresis easily identifies the mutations, since mutant allele generate smaller DNA fragments List of Restriction Enzyme PCR-RFLP How a restriction enzyme work? i.e. EcoRI PCR-RFLP http://tools.neb.com/NEBcutter2/index.php PCR-RFLP SNPicker Developed by Dr. Tianhua Niu and Dr. Zhenjun Hu 5. Agarose Gel Electrophoresis 5. Agarose Gel Electrophoresis 5. Agarose Gel Electrophoresis 5. Agarose Gel Electrophoresis 6. Genotype scoring Sample results of PCR-RFLP (CAPN10) 12 22 12 22 22 22 22 12 11 22 12 22 11 11 12 12 12 12 11 12 12 22 22 12 22 12 12 11 12 11 12 22 11 1 = major allele 2 = minor allele 11 Taqman SNP assay Taqman SNP assay 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. 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. 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. 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 Probe-Based Assay Chemistry How is Fluorescence Data Collected? Emission Filter Excitation Filter Light Source Detector How is Fluorescence Data Collected? Standard Procedures for Taqman SNP assay Taqman SNP assay The assays require only three components: 1. 2. 3. 1 to 20 ng of purified genomic DNA template SNP Genotyping Assay Mix (specific for each polymorphism) TaqMan Universal PCR Master Mix The assays require only one amplification step and an endpoint reading to obtain results. Default Taqman SNP program x40 cycles 95C 10min 92C 15sec 60C 1min SNP Genotyping Assay Mix Sequence-specific forward and reverse primers to amplify the SNP of interest 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 Genotype scoring Genotype scoring Compare PCR-RFLP and Taqman PCR-RFLP Taqman SNP assay Advantage Inexpensive for small setting studies Flexible for most kind of SNP genotyping Easy to perform Suitable for high throughput genotyping Need less information about target sequence Less prone to human error Faster, more efficient for SNP genotyping Disadvantage Need information about target sequence Not suitable for high throughput genotyping Prone to human error Not possible for all SNPs assays Higher expenses for equipment maintenance Limitations of Taqman Assay Not applicable for multi-allele SNPs No deletion No insertion No Microsatellites May not work if too many SNPs closed to the targeting site Other Molecular Technologies Direct Sequencing 1. 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. Other Molecular Technologies 1. Direct Sequencing (Continue) 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. Some form of DNA spotted on a chip (probes) Density of spots important One individual sample, many genotypes per assay Other Molecular Technologies 3. SBE (Single base extension) Allele specific primers designed polymerase synthezises the primer on the template exact one base before the SNP 4. Pyrosequencing short-read DNA sequencing and mutation/SNP analysis 5. SNPlex 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 Adaptation of method Many SNPs (>1000) few samples (< 600) Array platform Many genotypes per array Few arrays per day Few SNPs (< 100) many samples (> 600) Liquid based platform Multi-well plates provide flexibility Robotics can increase throughput Usually many samples/one SNP per plate Not true in “multiplex” situations Multiplexing vs. Pooling Multiplex Many genotypes from one reaction carried out in one tube Pooling Reactions carried out separately, analyzed together