* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Polymerase Chain Reaction
RNA polymerase II holoenzyme wikipedia , lookup
Gene expression wikipedia , lookup
Promoter (genetics) wikipedia , lookup
DNA sequencing wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Molecular evolution wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Biosynthesis wikipedia , lookup
Non-coding DNA wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Transformation (genetics) wikipedia , lookup
DNA supercoil wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Molecular cloning wikipedia , lookup
SNP genotyping wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Community fingerprinting wikipedia , lookup
Polymerase Chain Reaction • Introduction PCR: > background • Invented by Kary Mullis in 1985 • Noble Prize 1993 • Extremely powerful molecular biology technique DNA in the Cell chromosome cell nucleus Double stranded DNA molecule Target Region for PCR Individual nucleotides Practical applications of PCR 9 9 9 9 9 9 9 9 9 Amplify DNA for Cloning Modify DNA for Cloning Amplify DNA for sequencing without cloning DNA sequencing reaction Diagnose disease Pathogen screening Forensic analysis Paternity/maternity (relatedness) Population genetics (theoretical and applied) Polymerase Chain Reaction • How does it work? DNA Polymerization • A few things that you know but should keep in mind now. Nucleotide 1 = one strand of DNA Note polarity Nucleotide 2 Nucleotide 3 Nucleotide 4 P Nucleotide 1 S T one strand of DNA P Nucleotide 2 A S P Nucleotide 3 C S P Nucleotide 4 S G P 3’ S T S A Two strands of DNA •Double-stranded DNA •Anti-parallel •Hydrogen bonds •AT or GC pairs P P A T S S P P C G P C 3’ G S P S Polymerization of DNA occurs from 5‘ to 3‘ P S S 5’ 5’ DNA Polymerase Needs a Primer ss DNA 3´ + Nucleotides (dNTPs) + DNA polymerase = No DNA synthesis 5´ DNA Polymerase Needs a Primer primer ss DNA 5´ 3´ 3´ + Nucleotides (dNTPs) + DNA polymerase = NoDNA DNAsynthesis synthesis 5´ DNA Polymerase Needs a Primer primer ss DNA 5´ 3´ + Nucleotides (dNTPs) + DNA polymerase = DNA synthesis 5´ DNA Polymerization • How does nature do it? Extension - The Replication Fork 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ Primase Single strand binding proteins Laging Strand Okazaki fragment 5’ 3’ 5’ RNA Primers DNA Polymerase 5’ 3’ Helicase Leading Strand 5’ 3’ Functions And Their Associated Enzymes Function • Melting DNA Enzyme Helicase SSB Proteins Topisomerase • Polymerizing DNA DNA Polymerase • Providing primer Primase • Joining nicks Ligase PCR Purpose – Quickly make many copies of a region of a DNA molecule Method – Multiple rounds of DNA replication Components in PCR reaction – Target DNA, nucleotides, DNA polymerase, and primers Temperature cycling – DNA replication controlled by temperature… Temperature Cycling in PCR Temperature cycling – PCR process uses a machine (thermocycler) in which PCR reaction goes through ~30 cycles of three different temperature changes: ~95ºC – Melting temperature 50-65ºC – Annealing temperature 72ºC – Extension temperature Your polymerase must tolerate 95ºC ! Hot water bacteria: Thermus aquaticus Taq DNA polymerase Life at High Temperatures by Thomas D. Brock Biotechnology in Yellowstone © 1994 Yellowstone Association for Natural Science http://www.bact.wisc.edu/Bact303/b27 Polymerase chain reaction (PCR) analysis 1). primers are designed to flank the region to be amplified in target DNA 2). primers are annealed to denatured DNA 3). DNA is synthesized using Taq polymerase (from Thermus aquaticus) 4). primers are annealed again and the process is repeated through 20-30 cycles, geometrically amplifying the target sequence 5). DNA is analyzed by gel electrophoresis 1). 2). 3). 4). Copying DNA - the polymerase chain reaction Copying DNA - the polymerase chain reaction Copying DNA - the polymerase chain reaction Cells or tissue RT-PCR Reverse transcription PCR RNA isolation AAAAAAAA AAAAAAAA AAAAAAAA cDNA synthesis RNA-dependent DNA polymerase (reverse transcriptase) Gene specific priming AAAAAAAA Oligo(dT) priming AAAAAAAA Random priming AAAAAAAA TTTTTTTT AAAAAAAA AAAAAAAA AAAAAAAA TTTTTTT AAAAAAAA AAAAAAAA AAAAAAAA TTTTTTTTTT PCR amplification CONTAMINATION WITH GENOMIC DNA. DNAse treat RNA prior to experiment, use primers either side of introns, and do control reaction WITHOUT reverse transcriptase. Gene cloning • Why would you want to clone a gene or cDNA? – Sequence it – Express it and make the encoded protein (bacteria, mammalian cells, insect cells, yeast). – Make knockout/transgenic constructs to generate GM mice. • Popular methods for cloning PCR products include: – classical cloning that uses restriction enzymes and ligase (REAL) – TA Cloning® • PCR primers can be used to modify the ends of PCR products. – Add restriction sites. – Add sequence encoding epitope tags for recognition by standard Abs. Cloning your chosen gene Plasmids are circular, independent genetic elements found in bacterial cells. Put PCR product or DNA fragment into plasmid, and let bacteria do the amplification. CONTAINS: ORI – Origin of replication. Selectable marker Ampicillin resistance (Amp) ORI Amp PCR product/DNA fragment Transform Into E.coli ORI Amp Ampicillin containing plates Grow at 37°c In Ampicillin Lyse cells and Prepare DNA. Simple cloning methods. 1. Cloning using restriction enzymes – Design PCR primers with restriction enzyme (RE) sites – Amplify DNA – Digest with RE and ligate to vector digested with same enzyme CTCTGGATCCAGATATG AGAGACCTCGGTCTATAC 2. TA cloning BamHI CTCTG GAGACCTAG GATCCAGATATG GTCTATAC PCR • Practical issues. PCR COMPONENTS • TWO PRIMERS complimentary to “flanking” region of target – Forward strand – Reverse strand • Nucleotides – “building blocks” – A, T, C, G • DNA Polymerase – Taq (Thermus aquaticus) Lots of pipetting required PCR COMPONENTS • Master Mix – Primers – Reaction components – Dispensed into each tube for homogeneity between samples – Improves accuracy – Limit need for pipetting small volumes PCR CONTROLS • NEGATIVE CONTROL – PCR Master Mix ONLY – NO Template DNA • EXTRACTION BLANK – Extraction Reagents ONLY – NO Template DNA • POSITIVE CONTROL – Known positive sample – Plasmid DNA THERMAL CYCLER Thermal Cycling Temperatures 95 Temperature 95oC 95oC 72oC 60oC 95oC 72oC 60oC 95oC 72oC Typically 25-35 cycles operformed during PCR 60 C Single Cycle 30-40 cycles REAL TIME PCR USING SYBR GREEN 1 THE PROBLEM • NEED TO QUANTITATE DIFFERENCES IN mRNA EXPRESSION • SMALL AMOUNTS OF mRNA – SMALL AMOUNTS OF TISSUE – PRIMARY CELLS – PRECIOUS REAGENTS – JUST LOW EXPRESSION OF YOU’RE YOUR FAVROURITE GENE 2 THE PROBLEM • QUANTITATION OF mRNA – – – – northern blotting ribonuclease protection assay in situ hybridization PCR • • • • most sensitive can discriminate closely related mRNAs technically simple but difficult to get truly quantitative results using conventional PCR 3 NORTHERN control expt target gene internal control gene actin, GAPDH, RPLP0 etc Corrected fold increase = 10/2 = 5 Ratio target gene in experimental/control = fold change in target gene fold change in reference gene 4 Standards/Reference genes • same copy number in all cells • expressed in all cells • medium copy number advantageous – correction more accurate 5 Standards • The perfect standard does not exist 6 Standards • Commonly used standards – Glyceraldehyde-3-phosphate dehydrogenase mRNA – Beta-actin mRNA – MHC I (major histocompatability complex I) mRNA – Cyclophilin mRNA – mRNAs for certain ribosomal proteins • E.g. RPLP0 – 28S or 18S rRNA 7 REAL TIME PCR • kinetic approach • early stages • while still linear www.biorad.com 8 9 10 3. intensifier 1. halogen tungsten lamp 2b. emission filters 2a. excitation filters 5. ccd detector 350,000 pixels 4. sample plate 11 www.biorad.com CYCLE NUMBER 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 AMOUNT OF DNA 1 2 4 8 16 32 64 128 256 512 1,024 2,048 4,096 8,192 16,384 32,768 65,536 131,072 262,144 524,288 1,048,576 2,097,152 4,194,304 8,388,608 16,777,216 33,554,432 67,108,864 134,217,728 268,435,456 536,870,912 1,073,741,824 1,400,000,000 1,500,000,000 1,550,000,000 1,580,000,000 12 AMOUNT OF DNA 1600000000 1400000000 AMOUNT OF DNA 1 2 4 8 16 32 64 128 256 512 1,024 2,048 4,096 8,192 16,384 32,768 65,536 131,072 262,144 524,288 1,048,576 2,097,152 4,194,304 8,388,608 16,777,216 33,554,432 67,108,864 134,217,728 268,435,456 536,870,912 1,073,741,824 1,400,000,000 1,500,000,000 1,550,000,000 1,580,000,000 1200000000 1000000000 800000000 600000000 400000000 200000000 0 0 5 10 15 20 25 30 35 PCR CYCLE NUMBER A MOU N T OF D N A CYCLE NUMBER 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 10000000000 1000000000 100000000 10000000 1000000 100000 10000 1000 100 10 1 0 5 10 15 20 25 PCR CYCLE NUMBER 30 35 13 AMOUNT OF DNA 0 5 10 15 20 25 30 35 PCR CYCLE NUMBER 14 A MOU N T OF D N A 0 5 10 15 20 25 30 35 PCR CYCLE NUMBER 15 Linear ~20 to ~1500 16 Linear ~20 to ~1500 17 SERIES OF 10-FOLD DILUTIONS 18 SERIES OF 10-FOLD DILUTIONS 19 threshold Ct SERIES OF 10-FOLD DILUTIONS 20 threshold = 300 21 threshold 22 An example 23 control Δ Ct = target - ref RPLP0 con Δ Ct = 9.70 IL1-b con av =19.93 experiment av =29.63 Δ Ct = target - ref IL1-b vit Δ Ct = -1.7 RPLP0 vit av =18.03 av =19.80 Difference = ΔCt-ΔCt = ΔΔCt = 9.70-(-1.7) = 11.40 24 ΔΔCt = 11.40 for IL1-beta • 2 ΔΔCt variant: assumes efficiency is 100% Fold change = 211.40 = 2702 25 The melting curve: -d(RFU)/dT 26 OVERVIEW tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results 27 OVERVIEW tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results 28 IMPORTANCE OF RNA QUALITY • Should be free of protein (absorbance 260nm/280nm) • Should be undegraded (28S/18S ~2:1) • Should be free of DNA (DNAse treat) • Should be free of PCR inhibitors – Purification methods – Clean-up methods 29 OVERVIEW tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results 30 Importance of reverse transcriptase primers • Oligo (dt) • Random hexamer (NNNNNN) • Specific 31 REVERSE TRANSCRIPTION • adds a bias to the results • efficiency usually not known 32 OVERVIEW tissue extract RNA copy into cDNA (reverse transciptase) do real-time PCR analyze results 33 Importance of primers in PCR • • • • specific high efficiency no primer-dimers Ideally should not give a DNA signal – cross exon/exon boundary 34 EXON 1 EXON 1 INTRON 2 EXON 2 EXON 2 DNA RNA 35 How are you going to measure the PCR product • Directly – Sybr green – Quality of primers critical • Indirectly – In addition to primers, add a fluorescently labeled hybridization probe – Many different approaches to this, see Bustin J.Mol.Endocrinol. (2000) 25:169 36 Importance of controls • negative control (no DNA) – checks reagents for contamination • no reverse transcriptase control – detects if signal from contaminating DNA • positive control – checks that reagents and primers work – especially importance if trying to show absence of expression of a gene 37 Standards • same copy number in all cells • expressed in all cells • medium copy number advantageous – correction more accurate • reasonably large intron • no pseudogene • no alternate splicing in region you want to PCR 38 SPECIAL THANKS TO Christoph Schultes, who • Set up qRT-PCR in our lab • Made an overwhelming presentation on it (you saw about 50% of it). 39 PCR trouble shooting adapted from: http://publish.uwcm.ac.uk/study/medicine/dip_biomed_meth/ website_pcr.ppt CYCLING PARAMETERS Denaturation; 93°C - 95°C 30 secs – 1min Annealing; 37°C - 65°C Touchdown? 30 secs – 1min Extension; 72°C 1min (+ 30secs per 500bp DNA) 25-35 cycles Final extension 2-10mins PCR Agarose gel electrophoresis 3-4 hours The final product UV visualisation ALWAYS REMEMBER! PCR is a highly sensitive technique – contamination with unwanted DNA can be a problem Always run NEGATIVE controls Include a positive control if appropriate Use dedicated filtered tips and positive displacement pipettes Dedicated areas? Can use UV cabinets OPTIMISE PCR CONDITIONS AT THE OUTSET The specific method should be ROBUST Re-optimise for each set of primers X √ OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen • Thermo-stable DNA polymerase e.g. Taq polymerase • Oligonucleotides Design them well! • Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP) OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen • Thermo-stable DNA polymerase e.g. Taq polymerase • Oligonucleotides Design them well! • Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP) THE RAW MATERIAL Tissue, cells, blood, hair root, saliva, semen Obtain the best starting material you can. Some can contain inhibitors of PCR, so they must be removed e.g. Haem in blood Good quality genomic DNA if possible Blood – consider commercially available reagents Qiagen– expense? Empirically determine the amount to add OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen • Thermo-stable DNA polymerase e.g. Taq polymerase • Oligonucleotides Design them well! • Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP) CHOOSE YOUR POLYMERASE WITH CARE Number of options available Taq polymerase Pfu polymerase others •How big is the product? 100bp 40-50kb •What is end purpose of PCR? Sequencing - mutation detection Need high fidelity polymerase integral 3’ 5' proofreading exonuclease activity Cloning (TA cloning?) TA CLONING OF PCR PRODUCTS REQUIRES As A A PCR product Taq - yes T T pGEM-T pCR 2.1-TOPO Pfu - no OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen • Thermo-stable DNA polymerase e.g. Taq polymerase • Oligonucleotides Design them well! • Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP) PRIMER DESIGN IS VITAL •Length ~ 18-30nt (21nt) •Base composition; 50 - 60% GC rich pairs should have equivalent Tms Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C] •Initial use Tm–5°C •Avoid internal hairpin structures no secondary structure •Avoid A/T at the 3’ end •Avoid overlapping 3’ ends – will form primer dimers •Can modify 5’ ends to add restriction sites etc OPTIMISING PCR – THE REACTION COMPONENTS • Starting nucleic acid - DNA/RNA Tissue, cells, blood, hair root, saliva, semen • Thermo-stable DNA polymerase e.g.Taq polymerase • Oligonucleotides Design them well! • Buffer Tris-HCl (pH 7.6-8.0) Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP) TITRATE YOUR Mg2+ CONCENTRATION! 1 1.5 2 2.5 3 3.5 4 mM Normally, 1.5mM MgCl2 is optimal Best supplied as separate tube Always vortex thawed MgCl2 Mg2+ concentration will be affected by the amount of DNA, primers and nucleotides USE MASTERMIXES WHERE POSSIBLE Taken from http://info.med.yale.edu/genetics/ward/tavi/PCR.html “ALL BLOCKS AND TUBES ARE EQUAL BUT SOME ARE MORE EQUAL THAN OTHERS!” G. Orwell (not!) Taken from http://info.med.yale.edu/genetics/ward/tavi/PCR.html ADDITIVES? Depends on the PCR Can be used where products are diffuse or absent DMSO Formamide Glycerol Stratagene - Perfect Match http://taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html THE PERFECT RESULT Qiagen PCR methods If not………………………troubleshoot