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Eitan Friedman MD PhD Oncogenetics Unit Sheba Medical Center Tel-Hashomer

PCR: Polymerase Chain Reaction The basics and applications Sequence alterations: Mutations and polymorphisms. Eitan Friedman MD PhD Oncogenetics Unit Sheba Medical Center Tel-Hashomer. HOW PCR CAME ABOUT …. Modern PCR technology was initially developed

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Eitan Friedman MD PhD Oncogenetics Unit Sheba Medical Center Tel-Hashomer

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  1. PCR: Polymerase Chain Reaction The basics and applicationsSequence alterations: Mutations and polymorphisms Eitan Friedman MD PhD Oncogenetics Unit Sheba Medical Center Tel-Hashomer

  2. HOW PCR CAME ABOUT …. Modern PCR technology was initially developed by Dr.Kary B.Mullis, a research scientist At the Perkin-Elmer Cetus Corporation in April 1983 Driving up to Mendocino and thinking about an experiment to look at one particular letter of the genetic code, I designed a system in my mind. As I repaired the things I thought could go wrong with it, suddenlyI generated something that if I did it over and over again would be PCR. It would go 2, 4, 8, 16, 32 . . . in 30 cycles make as many base pairs from one little region as I had in the whole genome! That was the eureka point. I said: “holy shit! By putting the triphosphates [DNA building blocks] in there myself, I could do this process over and over and amplify the DNA…” Interview:Kary Mullis (april 1992)

  3. WHAT IS PCR ???????? • In vitro enzymatic synthesis and amplification of specific DNA sequences • rapid in vitro assay that mimics DNA replication that occurs in vivo a test tube system for DNA replication thatallows a “target “ DNA sequence to be selectively amplified, or enriched , several million-fold in just a few hours; a task that would have taken several days using recombinant technology Or Cellular DNA

  4. The PCR Process: How it Works • Chain reaction relies on DNA replication process • Repeated cycles of melting (strand separation), primer annealing, and primer extension by cycling temperatures • Need a tough enzyme to deal with high temperatures • Polymerases isolated from thermophilic bacteria (Thermus aquaticus, Pyrococcus furiosus) • The Process: (click to activate) http://vector.cshl.org/resources/BiologyAnimationLibrary.htm

  5. How does a PCR work?? DENATURATION 93°C - 95°C ANNEALING 37°C - 65°C 25-35 CYCLES DENATURATION 93°C - 95°C EXTENSION 72°C

  6. EACH PCR CYCLE HAS THREE STEPS • Denaturation; 93°C - 95°C 30 secs – 1min • Annealing; 37°C - 65°C30 secs – 1min depends on the melting temperature of duplex • Extension/Polymerisation; 72°C1min (+ 30secs per 500bp DNA) • 25-35 cycles • Final extension at 72°C for 2-10 minutes

  7. PCR 30x Melting Melting 100 94 oC 94 oC Extension Annealing Primers 72 oC Temperature 50 50 oC 0 T i m e 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’

  8. PCR Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ 5’ 3’

  9. PCR Melting 100 94 oC Temperature 50 0 T i m e 3’ 5’ Heat 5’ 3’

  10. PCR Melting Melting 100 94 oC 94 oC Extension Annealing Primers Temperature 72 oC 50 oC 50 0 T i m e 5’ 3’ 5’ 5’ 5’ 3’

  11. How many copies? • No target products are made until the third cycle. • The accumulation is not strictly a doubling at each cycle in the early phase. • At 30 cycles there are 1,073,741,764 target copies (~1109). • There are also 60 other DNA copies.

  12. How many cycles? • Increasing the cycle number above ~35 has little positive effect. • The plateau occurs when: • The reagents are depleted • The products re-anneal • The polymerase is damaged • Unwanted products accumulate.

  13. Thermal Cyclers • PCR cyclers available from many suppliers. • Many block formats and multi-block systems. • Reactions in tubes or 96-well micro-titre plates.

  14. UV Light Detection of PCR products Gel contains Ethidium Bromide, which binds to the DNA. When bound, the EtBr will fluoresce when exposed to UV light

  15. PCR Agarose gel electrophoresis 3-4 hours The final product UV visualisation

  16. Modifications and variations of PCR Multiplex PCR • PCR reactions can be devised in which several targets are amplified simultaneously — often used in diagnostic applications.

  17. APPLICATIONS OF PCR • Cloning of genes or gene fragmentssame species or homologous genes from different species (DOP-PCR) • Genetic diagnosis - Mutation detectionbasis for many techniques to detect gene mutations (sequencing) - 1/6 X 10-9 bp • Paternity testing • Mutagenesis to investigate protein function • Quantitate differences in gene expressionReverse transcription (RT)-PCR • Identify changes in expression of unknown genesDifferential display (DD)-PCR  • Forensic analysis at scene of crime • Industrial quality control

  18. Detecting pathogens using genome-specific primer pairs

  19. Identification of sequence alterations and genetic mutations

  20. Mutations and polymorphisms Mutation is a sequence alteration that is clearly associated with an abnormal phenotype, a disease state Polymorphism is a sequence alteration that can be found in more than 1% of the general population, and is not usually associated with a diseased phenotype

  21. Distinction between polymorphism and mutation • If the sequence alteration clearly affects the protein function in a deleterious manner – this is a mutation (e.g., truncation, nonsense mutation) • For missense mutations – either a bioassay, a protein model or segregation with the disease phenotype in families or the existence in the general population

  22. DNA polymorphisms • RFLPs • Co-dominant • Due to single base changes, insertions or deletions • VNTRs (minisatellites) • Probe detects repeat sequence (10-100 bp) • Microsatellite markers (di- & tri-NT repeats) • Probe detects flanking unique sequence • SNPs • Frequent and uniformly distributed in genome • Identified by sequencing

  23. Restriction fragment length polymorphism (RFLP) • a different kind of polymorphism that is useful for: • mapping genes (as with any genetic marker) • predicting those at risk for a disease • isolating genes by positional cloning

  24. Restriction fragment length polymorphisms (RFLPs) • Definitions • polymorphism • nucleotide sequence variation at allelic chromosomal sites • caused by base-pair mutation, deletion, or insertion... • RFLP • polymorphism that can be detected as a change in the • restriction fragment length pattern -- alleles are defined • by the sizes of bands obtained by Southern blot analysis • Two alleles ‘A’ and ‘a’ that differ • from one another by the absence • or presence of a restriction enzyme • cut site in the chromosome EcoRI EcoRI A GAGTTC EcoRI EcoRI EcoRI a GAATTC

  25. Southern blotting procedure human genomic DNA (isolated from many cells) - • gel electrophoresis • of the DNA fragments • gel will separate DNAs • according to size • restriction enzyme • digestion millions of DNA fragments + • hybridize membrane with • a 32P-labeled DNA probe • probe will base pair with the • complementary DNA strands • denature • DNA into • single- • strands • transfer • DNA fragments to • nitrocellulose membrane • expose membrane to X-ray film • develop film to visualize DNA band

  26. Fragments detected by Southern blotting • cut DNA by restriction enzyme digestion • hybridize labeled probe to DNA fragments • labeled probe will detect one restriction fragment as shown 32P • labeled hybridization probe • restriction fragment detected • by hybridization probe • adjacent fragment not detected

  27. polymorphism due to variable number of tandem repeats (VNTR): RFLP probe ‘A’, ‘B’, and ‘C’ are alleles 32P AA AB BC A B C polymorphism detected by Southern hybridization with a 32P- labeled probe polymorphism caused by deletion / insertion between restriction enzyme cut sites • VNTRs are useful because they are frequently highly polymorphic-- • there are more than two alleles, i.e., A,B,C,D,E… • highly polymorphic sites are more useful for distinguishing • chromosomes in the population because there are more • heterozygous chromosomes

  28. Inheritance of a hypervariable polymorphism Southern blot using a probe for a hypervariable VNTR Everyone in the pedigree is heterozygous at the locus

  29. Neuropsychiatric diseases caused by expansion • of trinucleotide repeats • Myotonic dystrophy • Fragile X syndrome • Spinal and bulbar muscular atrophy (Kennedy’s) • Huntington’s disease • Microsatellites • short regions of repeating DNA sequence in the genome • (because their G+C content is usually higher or lower • than the average for the genome they frequently appear • to band at a different buoyant density in CsCl gradients • and hence are called “satellites”) • microsatellites are often comprised of “trinucleotide repeats”

  30. Trinucleotide (or triple) repeats (a form of microsatellites) • the most common in human DNA are: • CAG, CGG, CAA, TAA, GAG • because DNA is double-stranded, the CAG repeat includes: • CAG, AGC, GCA, CTG, TGC, GCT • 5’CAGCAGCAGCAGCAG 3’ • 3’GTCGTCGTCGTCGTC 5’ • the number of repeats, and therefore their length at any • given locus, are polymorphic in the human genome • giving rise to VNTRs (variable number of tandem repeats)

  31. CAG CAG CAG CAG PCR product CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG Polymorphism at a triple repeat (VNTR) locus CAG CAG CAG CAG CAG

  32. Microsatellites (Simple Sequence Repeats) • Structure = Repeat (e.g., ga) Unique flanking regions • Number of repeats is highly variable among individuals Design primers ( ) complementary to flanking regions Amplify repeat region by polymerase chain reaction Analyze PCR products by polyacrylamide gel electrophoresis Marker is codominant and highly genetically informative

  33. Fluorescent Labeling of Microsatellites Enables Multiplexing • Acrylamide gel with 5 microsatellite loci and internal size standard • Four or more colors can be distinguished on many fluorimagers; coupled with size discrimination allows simultaneous analysis of nine or more loci

  34. Structure and inheritance of CTG repeats in myotonic dystrophy affected >75 premutation 45-75 myotonic protein kinase gene normal 5-30 (CTG)n

  35. Structure and inheritance of CGG repeats in fragile X syndrome affected >200 premutation 55-200 normal 6-55 FMR-1 gene (CGG)n

  36. CAG repeats in Huntington’s disease affected range: >39 repeats may or may not have disease: 36-39 repeats normal but can expand: 27-35 repeats normal range: 11-35 repeats (CAG)n (Gln)n

  37. SNP SNP-Single Nucleotide Polymorphisms . • SNPs are stable genetic markers, with a relatvely low mutation rate. • Typically, a common SNP is found about every 1000 bp. • In human, all combinations of substitution polymorphisms are observed, with A/G substitution SNPs being most prevalent.

  38. SNP

  39. SNPs are of interst for a variety of reasons: 1. A SNP which found in a functional gene region, may itself encode differences in protein form and expression. 2. SNPs may mark or track the presence of other genetic differences that cause phenotypes of interest. 3. They are useful in studying mutation rates and evolutionary history.

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