1POLYMERASE CHAIN REACTION AMPLIFIKASI FRAGMEN DNA Agustina Setiawati, MSc., Apt
2Background on the Polymerase Chain Reaction (PCR) Ability to generate identical high copy number DNAs made possible in the 1970s by recombinant DNA technology (i.e., cloning).Cloning DNA is time consuming and expensive (>>$15/sample).Probing libraries can be like hunting for a needle in a haystack.PCR, “discovered” in 1983 by Kary Mullis, enables the amplification (or duplication) of millions of copies of any DNA sequence with known flanking sequences.Requires only simple, inexpensive ingredients and a couple hours.DNA templatePrimers (anneal to flanking sequences)DNA polymerasedNTPsMg2+BufferCan be performed by hand or in a machine called a thermal cycler.1993: Nobel Prize for Chemistry
3The polymerase chain reaction (PCR) can selectively and rapidly amplify a given DNA sequence to large amountsUsed in cloning, sequencing, forensics, diagnosisSpecific primers hybridize on each side of the DNA sequence to be copiedEnzyme – Taq DNA polymerase – from Thermus aquaticus – resistant to high temperaturesVery sensitive – can amplify a sequence present in very low copy number
4How PCR works:Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence.Next, denature the DNA at 94˚C.Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA.Extend primers at 72˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus).Repeat the cycle of denaturing, annealing, and extension times to produce 1 million (220)to 35 trillion copies (245) of the target DNA.Extend the primers at 72˚C once more to allow incomplete extension products in the reaction mixture to extend completely.Cool to 4˚C and store or use amplified PCR product for analysis.
5Example thermal cycler protocol used in lab: Step 1 7 min at 94˚C Initial DenatureStep 2 45 cycles of:20 sec at 94˚C Denature20 sec at 64˚C Anneal1 min at 72˚C ExtensionStep 3 7 min at 72˚C Final ExtensionStep 4 Infinite hold at 4˚C StorageBIOL 362 samples processed in:MJ Research DNA Engine Dyad
12PCR – applications:1) The method of choice for cloning relatively short DNA sequences (under 10,000 nts) – can use to get genomic clone or cDNA clone2) Can detect infectious pathogens at very early stages3) PCR is used in forensic medicine to generate a DNA fingerprint – based on amplifying areas of the genome that contain VNTRs (variable number tandem repeats)
1310_29_PCR_viral.jpgUsing PCR to detect a viral genome in a drop of blood10_29_PCR_viral.jpg
1410_30_1_PCR_forensic.jpgPrimers are used to amplify areas with VNTRs, which differ in different chromosomes, different individuals10_30_1_PCR_forensic.jpg
1510_30_2_PCR_forensic.jpgThree areas amplified to generate a DNA fingerprint10_30_2_PCR_forensic.jpg
16Basic idea…lets say we want to amplify 1 gene of 500 bp from some bacterial DNA. * must know the sequence at the limits/ends of the DNA* design complementary primers anneal to template
1753533535Melt template, then rapidly cool* some primers will anneal to complementary sequence5335
185335Melt template, then rapidly cool* some primers will anneal to complementary sequence5335Add DNA polymerase* provide substrate + time to extend
195335Melt template, then rapidly cool* some primers will anneal to complementary sequence5335Add DNA polymerase* provide sunstrate + time to extend
20These 3 steps constitute 1PCR ‘cycle’, which will be repeated many times (usually 25-30) 1) melt template2) anneal oligonucleotide primers3) extend with DNA polymeraseIf ever confused about how PCR works…* draw out first three cycles25-30x
21Limitations – finite amounts of * dNTPs* primers* DNA polsExhaustion after 30
25Good Primer’s Characteristic A melting temperature (Tm) in the range of 52 C to 65 CAbsence of dimerization capabilityAbsence of significant hairpin formation (>3 bp)Lack of secondary priming sitesLow specific binding at the 3' end (ie. lower GC content to avoid mispriming)
26Uniqueness NOT UNIQUE! UNIQUE! There shall be one and only one target site in the template DNA where the primer binds, which means the primer sequence shall be unique in the template DNA.There shall be no annealing site in possible contaminant sources, such as human, rat, mouse, etc. (BLAST search against corresponding genome)Template DNA 5’...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3’TGCTAAGTTGCAGTCAACTGCTACTGCT AGTTGAPrimer candidate 1 5’-TGCTAAGTTG-3’NOT UNIQUE!Primer candidate 2 5’-CAGTCAACTGCTAC-3’UNIQUE!
27LengthPrimer length has effects on uniqueness and melting/annealing temperature. Roughly speaking, the longer the primer, the more chance that it’s unique; the longer the primer, the higher melting/annealing temperature.Generally speaking, the length of primer has to be at least 15 bases to ensure uniqueness. Usually, we pick primers of bases long. This range varies based on if you can find unique primers with appropriate annealing temperature within this range.
28PANJANG PRIMER Panjang primer 8 4 pangkat 8 = 65.536 pb Ukuran kromosom kb ada kemungkinan situsPanjang primer 17= pb diharapkan hanya menempel pada 1 situs
29Base CompositionBase composition affects hybridization specificity and melting/annealing temperature.Random base composition is preferred. We shall avoid long (A+T) and (G+C) rich region if possible.Template DNA 5’...TCAACTTAGCATGATCGGGCA...AAGATGCACGGGCCTGTACACAA...3’TGCCCGATCATGCTGCCCG CAT TT AT GCUsually, average (G+C) content around 50-60% will give us the right melting/annealing temperature for ordinary PCR reactions, and will give appropriate hybridization stability. However, melting/annealing temperature and hybridization stability are affected by other factors. Therefore, (G+C) content is allowed to change.
30Melting TemperatureMelting Temperature, Tm – the temperature at which half the DNA strands are single stranded and half are double-stranded.. Tm is characteristics of the DNA composition; Higher G+C content DNA has a higher Tm due to more H bonds.CalculationShorter than 13: Tm= (wA+xT) * 2 + (yG+zC) * 4Longer than 13: Tm= *(yG+zC-16.4) /(wA+xT+yG+zC) (Formulae are from
31Annealing Temperature Annealing Temperature, Tanneal – the temperature at which primers anneal to the template DNA. It can be calculated from Tm .Tanneal = Tm_primer – 4C
32Primer Pair MatchingPrimers work in pairs – forward primer and reverse primer. Since they are used in the same PCR reaction, it shall be ensured that the PCR condition is suitable for both of them.One critical feature is their annealing temperatures, which shall be compatible with each other. The maximum difference allowed is 3 C. The closer their Tanneal are, the better.
33Summary ~ when is a “primer” a primer? 5’3’5’3’5’3’3’5’
34Summary ~ Primer Design Criteria Uniqueness: ensure correct priming site;Length: bases.This range varies;Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible;Optimize base pairing: it’s critical that the stability at 5’ end be high and the stability at 3’ end be relatively low to minimize false priming.Melting Tm between C are preferred;Assure that primers at a set have annealing Tm within 2 – 3 C of each other.Minimize internal secondary structure: hairpins and dimmers shall be avoided.
36RT-PCR Procedure mRNA was isolated from a tissue cell culture Reverse transcriptase used to synthesize cDNA from mRNAPCR performed on cDNA using Taq polymerase and primers specific for Syk geneBeta-actin used as an external control for the RT- PCRSouthern blot used a biotynlated internal Syk probe used to confirm amplification of Syk mRNA
37RT-PCR ResultsSyk is expressed in normal mammary gland tissue and breast epithelial cellsSeveral carcinoma cell lines expressed Syk stronglySome carcinoma cell lines poorly expressed SykSome carcinoma cell lines completely lack Syk expressionMiddle part of upper figure is the Southern Blot – confirms amplification of Syk mRNABeta-actin used as a control – ubiquitously expressed in culturesPCR products also sequenced to confirm amplification of Syk mRNA
38Advantages Disadvantages RT-PCR is much faster and more sensitive that RNase protection assaysRT-PCR is very sensitive and can detect low levels of mRNA in cellsRT-PCR requires a lot of preparation and must be strictly controlledNon-competitive RT-PCR may result in false conclusions because experimental results are caused by differences in PCR conditions
39REAL TIME PCRtitle page for 7700, quantitative PCR presentationRT PCR2
40Apakah Real-Time PCR ?‘Real Time’ dapat dikatakan sebagai mengoleksi dan menganalisa data yang terjadi selama proses reaksi‘Real Time PCR’ berarti amplifikasi dan analisa terjadi bersamaanDikenal sebagai ‘Rapid Cycle PCR’ dengan siklus temperatur antara detikProduk PCR dapat dianalisa selama proses amplifikasiMenggunakan ‘pewarna DNA’ dan ‘probe fluoresensi’Data dikoleksi dari tabung yang sama didalam instrumen yang samaTidak ada transfer sampel, penambahan reagensia atau gel separasi
41Apakah Real-Time PCR ? Format deteksi : SYBR Green IHybridization Probes (HybProbe Probes)Hydrolysis probes / Taqman ProbesSimpleProbe ProbesDapat melakukan ‘real-time quantitative PCR’‘Real Time PCR’ adalah metode yang ‘powerful’, sederhana dan cepat
42Applications For Detecting and Quantifying Transcripts Quantifying virusesPathogen detectionGene expressionDrug therapyDNA damageImmune responseGenotyping
43Monitoring PCR Reaction Agarose Gel BlottingLightCycler
44What’s Wrong With Agarose Gels? Poor precision.Low sensitivity.Short dynamic range < 2 logs.Low resolution.Non-automated.Size-based discrimination only.Results are not expressed as numbers.Ethidium bromide staining is not very quantitative.
45Approaches in quantifying by PCR .3. Real-time RT-PCR (QPCR). Measurement occurs during exponential phase..
46Available Chemistries for detecting PCR product (amplicon). 1. Intercalating dyes that fluoresce, e.g. SYBR Green I.2. Hybridization probes, Scorpions. Molecular Beacons. Donor probe excites acceptor by FRET.3. Hydrolysis probes, Quench is by non-radiative transfer: Taqman system.4. Simple probes
471. SYBR Green IKetika SYBR Green I berikatan dengan dsDNA, akan terjadi peningkatan fluoresensiSelama tahapan PCR yang berbeda, intensitas dari sinyal fluoresensi akan berbeda, tergantung dari jumlah dsDNA yang ada
502. HybProbe ProbesHybridization probe merubah fluoresensi pada saat hibridisasi dengan ‘fluorescene resonance energy transfer (FRET)’
51Fluorescence Resonance Energy Transfer (FRET) In addition to the 5’ nuclease activity, TaqMan utilizes FRET or energy transfer. The 5’ reporter dye has a higher energy than the 3’ quencher dye. When the probe is intact, energy from the 5’ dye is transferred (energy flows down hill) to the 3’ dye. Thus, the intact probe has a low emission from the 5’ dye and high emission from the 3’ dye.When the probe is cleaved, the distance between the two free dyes increases to the extent that the energy transfer cannot occur. Thus, the 5’ dye now has a greater emission than the 3’ dye.
52FRET is Nonradiative energy transfer FRET occurs by a nonradiative dipole-induced dipole interaction.
533. Taqman probes are “hydrolysis” probes PolymerizationTaqMan® ProbeForwardPrimerRQ5’3’5’5’3’5’ReversePrimer
574. Simple ProbesSimple Probe adalah bentuk sederhana dari hybridization probe dan hanya menggunakan 1 probe sajaKetika terjadi hibridisasi akan memancarkan sinyal fluoresensi yang lebih besarPerubahan sinyal fluoresensi tergantung dari status hibridasi dari probe, semakin stabil hibridisasinya semakin tinggi temperatur meltingUntuk aplikasi SNP genotyping dan deteksi mutasi
58Molecular Beacons are “hybridization” probes STEM-LOOP DESIGN MAY HAVE LOWER BACKGROUND FLUORESCENCE MEASURED DURING ANNEALING STEP, DISSOCIATES DURING EXTENSION,BEFORE PROBES ARE DISPLACED BY POLYMERASE