PCR. What is PCR? What is PCR? Why chain? What is PCR? Why Polymerase?

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1 What is PCR? PCR the swiss army knife Claudia Stäubert, Institute for biochemistry PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro. It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in Sep 17, 2010 What is PCR? Why Polymerase? It is called polymerase because the only enzyme used in this reaction is DNA polymerase. What is PCR? Why chain? It is called chain because the products of the first reaction become substrates of the following one, and so on. What is PCR? The reaction components 1) Target DNA - contains the sequence to be amplified. What is PCR? The reaction 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dntps - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg ++ ions - cofactor of the enzyme 6) Buffer solution maintains ph and ionic strength of the reaction solution suitable for the activity of the enzyme PCR tube THERMOCYCLER 1

2 How PCR works PCR swiss army knife Allele-specific PCR (SNP detection) Assembly PCR (artificial synthesis of long DNA sequences) Asymmetric PCR (preferential amplification of one DNA strand) Helicase-dependent amplification (usage of constant temperture no cycling) Hot-start PCR (reduction of non-specific amplification) Intersequence-specific PCR (ISSR) (DNA fingerprinting) Inverse PCR (used to identify flanking sequences around genomic inserts) Ligation-mediated PCR (small DNA-linkers ligated to DNA, multiple primer annealing to linkers) e.g. genome walking PCR swiss army knife Methylation-specific PCR (MSP) (for detection of CpG islands in genomic DNA) Miniprimer PCR (thermostable polymerase that can extent smalligos 9-10bp) Multiplex Ligation-dependent Probe Amplification (MLPA) (amplification of multiple targets with a single primer pair) Multiplex PCR (multiple primer sets within a single PCR reaction prodution of amplicons of varying size) Nested PCR (increased specifity of amplification, 2 primer sets in 2 successive PCRs) Overlap-extension PCR (genetic engineering technique insertion of alterations) PCR swiss army knife Quantitative PCR (measurement of quantity of a PCR product in real-time using fluorescent dyes or fluorophore containing DNA probes) PCR swiss army knife Random-mutagenesis PCR (introduces randonly mutations) Reverse Transcription PCR (amplification of DNA from RNA using reverse transcriptase) Site-directed mutagenesis PCR (introduces mutations at specific sites) Solid phase PCR (either primers or probes are covalently linked) Thermal asymmetric interlaced PCR (TAIL PCR) (isolation of unknown sequence flanking a known sequence) Touchdown PCR (gradual reduction of annealing temerature as PCR cycling progresses) DNA fingerprinting (forensics) 2

3 Sequencing Mammalian/human genomes ancestral genomes (Neanderthal) biomolecular computing Taxonomic relationships/phylogenies Genetic engineering Molecular cloning Site-directed/random mutagenisis Gene expression studies (RNA) Classification of organism DNA fingerprinting (forensics) Relation to other species Similarities Differences BUT what to do when you have only Fossils Trace amounts small organisms? Classification of organism Classification of organism SEQUENCING 3

4 PCR-based locus specific RFLP Classification of organsims specific locus amplified by PCR PCR product cut by restriction endonucleases variation in fragment length detectable by gel electrophoresis REP-PCR repetitive extragenic palindromic (REP) elements: 38bp sequence, 6 degenerate positions and 5bp variable loop enterobacterial repetitive intergenic consensus (ERIC): 126bp elemts described for enteric bacteria amplification patterns used to discriminate between strains RAPD assay random amplified polymorphic DNA (RAPD) short (9-10bp) random sequence primer number and location of random primer sites vary for different strains of bacteria specific pattern on gel CFLP cleavase fragment length polymorphism (CFLP) Cleavase I: thermostable, engineered endonuclease, recognizes structures formed by base-paired DNA at elevated temperatures 95 C 60 C single strand DNA selfbase pairing phase unique hairpin structures cut by Cleavase I unique pattern on gel AFLP amplified fragment lenghth polymorphism (AFLP) genome fingerprinting technique digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments. selective amplification of a subset of these DNA fragments electrophoreses analyses of specific band pattern 4

5 AFLP DNA fingerprinting (forensics) Molecular archaeology use DNA or mtdna (mitochondrial DNA) to determine species of natural archaeological finds as well as determine blood lines and/or sex of animal or human remains reconstruction of subsistence and related cultural activities possible DNA fingerprinting (forensics) Molecular epidemiology Detection of pathogenes A science that focuses on the contribution of potential genetic and environmental risk factors, identified at the molecular level, to the etiology, distribution and prevention of disease within families and across populations" Descriptive and analytical studies to evaluate host/environmental interactions in disease Development of prevention strategies for the control of bacterial, parasitic and viral disorders through molecular diagnosis Prevention of non-communicable diseases and genetic disorders by assessing risk and identifying susceptible individuals through genetic screening 5

6 DNA fingerprinting (forensics) Molecular ecology Field of evolutionary biology that is concerned with applying molecular population genetics, molecular phylogenetics, and more recently genomics to traditional ecological questions (e.g., species diagnosis, conservation and assessment of biodiversity, species-area relationships, and many questions in behavioral ecology). DNA fingerprinting (forensics, paternity testing) DNA fingerprinting (DNA typing/profiling) Even though two unrelated humans differ in their DNA only by 0.1 to 0.2% there are still up to 6 million basepair differences It is these differences that are used to create a unique DNA fingerprint also known as DNA profile Human genetics variation Primarily two types of genetic mutation events create all forms of variations: Single base mutation which substitutes one nucleotide for another Single Nucleotide Polymorphisms (SNP) Insertion or deletion of one or more nucleotide(s) Tandem Repeat Polymorphisms Insertion/Deletion Polymorphisms 4 criteria for selecting useful DNA fingerprinting markers 1. Markers should be polymorphic (so that they are informative) 2. Markers should be single locus (so that they occur in only one location in the genome and there is no ambiguity about their number or position) 3. Markers should be located on different chromosomes (so that the markers are independent) 4. For some applications such as the study of population size and changes over time, markers should be neutral (so that they are not correlated with selection or adaptation; unless selection of adaptation are to be studied; selection confounds estimates of population size parameters) 6

7 Good DNA markers Minisatellite repeat (VNTR) Repeated DNA Minisatellites (VNTRs = variable number tandem repeats) Repeated units (5-50 times) of 10 to several 100 bp GC-rich inherited codominantly Microsatellites (STRs = short tandem repeats) Repeated units of 2-6 bp One Mechanism of VNTR Creation PCR amplification of VNTR This process requires primers that anneal just outside the VNTR Single-Locus VNTR Multi-Locus VNTR Single-locus minisatellite VNTRs generates at most two bands Though not as unique as multi-locus VNTRs they are simple to use Multiple single-locus VNTRs are used to give a DNA fingerprint 7

8 Microsatellites Strand slippage replication at STR short tandem repeats (STR) or simple sequence repeat (SSR) Heterozygote Male 5 -TAATAATAATAATAATAATAA----3 Female 5 -TAATAATAATAATAATAATAATAA-3 Homozygote Male 5 -TAATAATAATAATAA-3 Female 5 -TAATAATAATAATAA-3 One proposed explanation for their fast rate of evolution is slippage during DNA replication. Microsatellites (STR) 7 repeats 8 repeats The number of repeat units varies between alleles while the PCR primers bind to conserved flanking regions Homozygote = both alleles are the same length Heterozygote = alleles differ and are resolved from one another STR Multiplexing 16 or more loci can be amplified at once Sensitivities to levels less than 1 ng of DNA Ability to handle mixtures and degraded samples Different fluorescent dyes used to distinguish STR loci with overlapping allele size ranges DNA profiling DNA Evidence Can Exonerate As Well As Implicate 8

9 DNA fingerprinting (forensics, paternity testing) Single nucleotide polymorphisms (SNPs) Single base pair differences occur about once every bp. In most populations there is a common SNP, and several less common SNPs. About 3 million SNPs occur in the human genome, and these are becoming popular genetic markers. SNPs can be used just like other genotyping markers, but more loci typically must be used because only 4 alleles (G, G, C, T) are possible. Typing a SNP DNA fingerprinting (forensics, paternity testing) Mutation detection Most mutations involve single nucleotide polymorphisms (SNPs) PCR amplifications with one primer binding to site of SNP Only properly matching primer gives product Use one primer/reaction for each known allele Mutation detection Huntington s disease is an example of a microsatellite triplet repeat in a coding region autosomal-dominant 9

10 DNA fingerprinting (forensics) Prenatal Diagnosis of Genetic Diseases Cells from amniocentesis Possible carriers by buccal swab Analyze relevant loci Molecular analysis of a family with an autosomal recessive disease 644 bp 440 bp 204 bp Sequencing Biomolecular computing Bioinformatics Mammalian/human genomes ancestral genomes (Neanderthal) biomolecular computing Taxonomic relationships/phylogenies Sequencing Taxonomic relationships/phylogenies Bioinformatics Mammalian/human genomes ancestral genomes (Neanderthal) biomolecular computing Taxonomic relationships/phylogenies Krause et al

11 Genetic engineering Summary Molecular cloning Site-directed mutagenisis Gene expression studies blood, chorionic villus, amniotic fluid, semen, hair root, saliva, microorganisms, fossils, Gel Analysis, Restriction Digestion, Sequencing, Cloning Conclusion The speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications. Quick Quiz The purpose of PCR is to: A. make copies of a specific region of DNA B. identify the presence of particular genes C. make sufficient genetic material for future molecular work D. all of the above Quick Quiz Quick Quiz A thermocycler protocol is comprised of which series of steps? A. Decontamination, amplification, excision B. Annealing, polymerization, cooling C. Transcription, translation, expression D. Denaturing, annealing, extension PCR amplifies molecular products in a(n) fashion: A. Logarithmic B. Exponential C. Linear D. Random 11

12 Quick Quiz Primers: A. Stabilize double stranded DNA B. Are enzymes that catalyze the copying process C. Are short single-stranded DNA fragments D. Are long strings of nucleotides Quick Quiz Which of the following reagents is NOT in a master mix? A. MgCl 2 B. Template DNA C. H 2 O D. dntps Quick Quiz If you forgot to add one of your primers your resultant gel will probably have A. No bands B. A smear C. A band of the wrong size D. Many bands 12