Direct and Indirect Mutation Analysis I - PCR PDF
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NEU Faculty of Pharmacy
Merdiye Mavis
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Summary
This document is a presentation or lecture on polymerase chain reaction (PCR). It covers various aspects of PCR, including its application in the detection of mutations, different types of PCR, and steps involved in the PCR process. It explores the analysis of mutations through direct and indirect approaches. The document also discusses reagents, equipment, and how to analyze PCR products.
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Direct and indirect mutation analysis I: PCR Research Assist. Merdiye Mavis Mutation Detection The detection of mutations has an increasingly central role in various areas of genetic diagnosis – Preimplantation genetic diagnosis (PGD) – Prenatal diagnosis (PND) – P...
Direct and indirect mutation analysis I: PCR Research Assist. Merdiye Mavis Mutation Detection The detection of mutations has an increasingly central role in various areas of genetic diagnosis – Preimplantation genetic diagnosis (PGD) – Prenatal diagnosis (PND) – Presymptomatic testing – Confirmational diagnosis – Forensic/identity testing. Mutation Detection There are two approaches for genetic diagnosis: – Direct: diagnosis essentially depends on the detection of the genetic variations responsible for the disease. – Indirect: depends on the results from a genetic linkage analysis using DNA markers such as STR (short tandem repeat) or VNTR (variable number tandem repeat) markers flanking or within the gene Two groups of tests, molecular and cytogenetic, are used in genetic syndromes Cytogenetics and Molecular Cytogenetics Conventional Karyotyping Fluorescence in situ hybridization (FISH) Comperative genomic hybridization (CGH) Molecular Diagnostics It combines laboratory medicine with molecular genetics to develop DNA/RNA-based analytical methods for monitoring human pathologies. Known Mutations – Polymerase chain reaction (PCR) and its versions Unknown Mutations – Single Strand Conformational Polymorphism (SSCP) – Denaturing Gradient Gel Electrophoresis (DGGE) – Heteroduplex analysis – Restriction Length Polymorphism (RFLP) Polymerase Chain Reaction Discovered in 1983 by Kary Mullis 1993: Nobel Prize for Chemistry Polymerase chain reaction (PCR) In vitro version of DNA Replication Provides multiple copies of specific DNA sequence – ‘Molecular photocopying’ DNA Replication in Cells Stages in PCR 3’ 5’ 1. Denaturation – Heat to separate 5’ 3’ double strands – This occurs at 94-95 ºC – Mimics the function of 5’ 3’ helicase in the cell. 3’ 5’ Stages in PCR 5’ 3’ 2. Annealing – Occurs at 54 ºC (50-65 ºC) Primer – Primers bind to template DNA sequence hybridization of primers to Primer the DNA sequence – Primers: 3’ 5’ one is complimentary to the one strand at one end of the target sequence other is complimentary to the other strand at the other end of the target sequence Stages in PCR 3. Elongation 5’ 3’ – Primer is extended with addition of dNTPs with Taq 3’ 5’ polymerase – The extension of the strand in 5’ 3’ the 5’ -3’ direction starting at the primers attaching the appropriate nucleotide 3’ 5’ – Temperature depends on the polymerase used. – Taq polymerase has an maximum activity at 75-80 ºC. – When Taq polymerase is used elongation step takes place at 72 ºC. PCR Amplification PCR Components dNTPS (bases) MgCl2 PCR Primers Primers – Short synthetic oligonucleotide – ssDNA sequences flanking region of interest – Present in excess – up to 0.5mM (too high – primer dimers) Primer Design for PCR Primer Length: Optimal length of PCR primer is 18-22 bp Long enough for adequate specificity Short enough for primers to bind easily to the template at the annealing temperature. Primer Melting Temperature (Tm) the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability Optimal Tm : 52-58 oC GC content of the sequence gives a fair indication of the primer Tm Tm=2X(A+T)+ 4X(C+G) Primer Design for PCR Primer Annealing Temperature the primer melting temperature is the estimate of the DNA-DNA hybrid stability and critical in determining the annealing temperature. Too high Ta will produce insufficient primer-template hybridization resulting in low PCR product yield. Too low Ta may possibly lead to non-specific products caused by a high number of base pair mismatches,. GC Content: The GC content of primer should be 40-60%. Primer-Dimer Results from primers annealing each other at 3’ ends. Extended primers are no longer available to prime target for PCR. atcggactatcga gctatacttatggcca atcggactatcgatatgaataccgga tagcctgatagctatacttatggcca Reagents for PCR DNA polymerase Enzyme responsible for copying the sequence starting at the primer from the single DNA strand by adding nucleoside triphosphates to the 3’ end of the growing strand DNA polymerase uses each strand as a template to synthesize new strands of DNA, complementary order of nucleotides. This enzyme is heat-tolerant thermally tolerant (survives the melting temperature of DNA denaturation) the process is more specific, higher temperatures result in less mismatch – more specific replication Many types available Some modified to allow hot start Some have long half life / stable at high temperature Some have high rate of processivity Reagents for PCR MgCl2 Is required for enzyme activation and amplification It stabilizes dsDNA and raises the Tm. Mg2+ concentration controls the specificity of the reaction. Reagents for PCR Buffer - providing a suitable chemical environment for optimum activity and stability of the DNA polymerase dNTPs (bases) PCR - before the thermocycler 95º C 55º C 72º C 5 min 3 min 5 min 35 times 8 hours per PCR! Thermocycler PCR tube with all the reagents THERMOCYCLER The thermal cycler allows heating and cooling of the reaction tubes to control the temperature required at each reaction step. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. How do we analyse the PCR products? Gel electrophoresis Sequencing Fluorescent PCR Blotting Short tandem repeat (STR) analysis Gel electrophoresis Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. Equipment Needed for gel electrophoresis work – Box to hold the gel – Comb to create small wells in the agarose gel to put the DNA sample in – Positive and negative electrodes to create the electrical current – Power supply – Gel photo imaging system Jel Elektroforezi Gel Electrophoresis Fragmentation products of differing length are separated Separation of DNA fragments Separation based (mostly) on length – longer molecules move slower. How does gel electrophoresis work? DNA is forced by an electrical current through a firm gel – Phosphate group in DNA is negatively charged so it is moved towards a positive electrode by the current – Longer fragments have more nucleotides Have a larger molecular weight Are bigger in size So aren’t able to pass through the small holes in the gel and get hung up at the beginning of the gel – Shorter fragments are able to pass through and move farther along the gel – Fragments of intermediate length travel to about the middle of the gel How does gel electrophoresis work? DNA fragments are then visualized in the gel with a special dye Ethidium Bromide is commonly used The number of nucleotides are then estimated by comparing it to a known sample of DNA fragments which is run through the gel at the same time DNA ladder Sequencing DNA sequencing = determining the nucleotide sequence of DNA Dideoxy sequencing developed by Frederick Sanger in the 1970s. Sanger sequencing or chain termination method 1980: Walter Gilbert (Biol. Labs) & Frederick Sanger (MRC Labs) Sequencing 1. The double stranded DNA molecule to be sequenced is denatured in a solution of NaOH. 2. One of theses strands of DNA can be chosen for sequencing. 3. The solution containing the single strand of DNA is mixed with: A labeled DNA primer (complementary to the 3’ end of the strand to be sequenced) DNA Polymerase 4 types of deoxynucleoside 5’-triphosphates - dNTPs (dATP, dTTP, dCTP, and dGTP) - bases A tiny quantity of a specific labeled dideoxynucleoside triphosphates - ddNTPs (ddATP, ddTTP, ddCTP, or ddGTP) 4. The reaction mixture is prepared 4 times, each tube containing different types of ddNTPs. ddNTPs ddNTPs are added to each of the four reaction tubes at 1/100th the concentration of normal dNTPs and in the reaction mixture they compete with dNTPs. ddNTP’s are chain-elongation inhibtors of DNA polymerase. ddNTPs possess a 3’-H instead of 3’-OH. The absence of the 3'-hydroxyl group means that, after being added by a DNA polymerase to a growing nucleotide chain, no further nucleotides can be added as no phosphodiester bond can be create – lead to chain termination Sequencing Whenever the labeled ddNTPs are incorporated in the chain, DNA synthesis terminates. Each of the four reaction mixtures produces a population of DNA molecules with DNA chains terminating at all possible positions. Extension products in each of the four reaction mixtures also end with a different labeled ddNTP (depending on the base). Sequencing Sequencing Polyacrylamide gels Each reaction mixture is loaded on wells of the polyacrylamide gel and is electrophoresed on a polyacrylamide gel or analysed by genetic analyser. Pattern of bands in each of the four lanes is visualized on X-ray film. Location of “bands” in each of the four lanes indicate the size of the fragment terminating with a respective labeled ddNTP. DNA sequence is deduced from the pattern of bands in the 4 lanes. Automated DNA Sequencing 1. Automated DNA sequencing uses ddNTPs labeled with fluorescent dyes. 2. Combine 4 dyes fluorescing at different wavelengths in one reaction tube and electrophores in one lane on a capillary containing polyacrylamide. 3. Capillary is thinner then gel higher voltage even faster. 4. UV laser detects dyes and reads the sequence. 5. Sequence data is displayed as colored peaks (chromatograms) that correspond to the position of each nucleotide in the sequence. 6. Throughput is high, up to 1200 bp per reaction and 96 reactions every 3 hours with capillary sequencers. Automated DNA Sequencing Applied Biosystems PRISM 3700 (Capillary, 96 capillaries) Applied Biosystems PRISM 3100 (Capillary, 16 capillaries) Automated DNA Sequencing Trace files (dye signals) are analyzed and bases called to create chromatograms. Different Types of PCR Reverse Transcription PCR (RT-PCR) To detect RNA expression Qualitatively detect gene expression through creation of complementry DNA (cDNA) Is used to clone expressed genes by reverse transcribing the RNA of interest into its DNA complement through the use of reverse transcriptase. Rnase H enzyme degrades RNA. The newly synthesized cDNA is amplified using traditional PCR. RT-PCR The amplified DNA fragments that are produced can be analysed by agarose gel electrophoresis or fluorescent PCR or real time PCR. The amount of amplified fragment produced is proportional to the amount of target mRNA in the original RNA sample. RT-PCR is extremely sensitive and can be used to detect very rare mRNA species. Allele-specific PCR Allele-specific PCR is used to identify or utilize single nucleotide polymorphisms (SNPs: single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles. Uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence. Nested PCR Nested PCR increases the specificity of DNA amplification by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCR. – In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. – The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences. Methylation-specific PCR (MSP): MSP is used to detect methylation of CpG islands in genomic DNA. 1- DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. 2- Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. – At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA – One set recognizes DNA with uracil to amplify unmethylated DNA. Real-time quantitative PCR: Same as PCR, but measures the abundance of DNA /PCR product as it is amplified. Quantitatively measures starting amounts of DNA, cDNA or RNA in a sample. Can also be used to quantitatively estimate fraction of DNA from various organisms in a heterogenous sample (e.g, can be used to measure abundance of different microbes in soil sample). Commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Uses reverse transcriptase to generate cDNA for the template. Two common methods for the detection of PCR products in real-time PCR are 1. Non-specific fluorescent dyes that intercalate with any double-stranded DNA Fluorescent dyes, such as SYBR Green , EvaGreen 2. Sequence-specific DNA probes consisting of oligonucleotides labelled with a fluorescent reporter Allows detection only after hybridization of the probe with its complementary sequence Fluorophore-containing DNA probes such as TaqMan SYBR Green (Yeşil) SYBR Green fluoresces strongly when bound to DNA, but emits little fluorescence when not bound to DNA. SYBR Green (Yeşil) SYBR Green fluorescence is proportional to the amount of DNA amplified, detected with a laser or other device. Real-time quantitative PCR amplification plot: Experimental samples are compared to control sample with known concentration of cDNA. PCR has become a very powerful tool in molecular biology One can start with a single sperm cell or stand of hair and amplify the DNA sufficiently to allow for DNA analysis. One can amplify fragments of interest in an organism’s DNA by choosing the right primers. One can use the selectivity of the primers to identify the likelihood of an individual carrying a particular allele of a gene. PCR and Disease Primers can be created that will only bind and amplify certain alleles of genes or mutations of genes This is the basis of diagnostic tests and genetic counseling PCR is used for diagnosis of genetic diseases. Some diseases that can be diagnosed with the help of PCR: Huntington's disease Cystic fibrosis Human immunodeficiency virus