Background 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 template Primers (anneal to flanking sequences) DNA polymerase dNTPs Mg 2+ Buffer Can be performed by hand or in a machine called a thermal cycler. 1993: Nobel Prize for Chemistry
The polymerase chain reaction (PCR) can selectively and rapidly amplify a given DNA sequence to large amounts Used in cloning, sequencing, forensics, diagnosis Specific primers hybridize on each side of the DNA sequence to be copied Enzyme – Taq DNA polymerase – from Thermus aquaticus – resistant to high temperatures Very sensitive – can amplify a sequence present in very low copy number
How PCR works: 1.Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence. 2.Next, denature the DNA at 94˚C. 3.Rapidly cool the DNA (37-65˚C) and anneal primers to complementary s.s. sequences flanking the target DNA. 4.Extend primers at 72˚C using a heat-resistant DNA polymerase (e.g., Taq polymerase derived from Thermus aquaticus). 5.Repeat the cycle of denaturing, annealing, and extension times to produce 1 million (2 20 )to 35 trillion copies (2 45 ) of the target DNA. 6.Extend the primers at 72˚C once more to allow incomplete extension products in the reaction mixture to extend completely. 7. Cool to 4˚C and store or use amplified PCR product for analysis.
Example thermal cycler protocol used in lab: Step 17 min at 94˚CInitial Denature Step 245 cycles of: 20 sec at 94˚CDenature 20 sec at 64˚CAnneal 1 min at 72˚CExtension Step 37 min at 72˚CFinal Extension Step 4Infinite hold at 4˚CStorage BIOL 362 samples processed in: MJ Research DNA Engine Dyad
10_27_1_PCR_amplify.jpg The polymerase chain reaction – used to amplify a specific DNA sequence with cyclical changes in temperature
PCR – applications: 1) The method of choice for cloning relatively short DNA sequences (under 10,000 nts) – can use to get genomic clone or cDNA clone
PCR – applications: 1) The method of choice for cloning relatively short DNA sequences (under 10,000 nts) – can use to get genomic clone or cDNA clone 2) Can detect infectious pathogens at very early stages 3) 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)
10_29_PCR_viral.jpg Using PCR to detect a viral genome in a drop of blood
10_30_1_PCR_forensic.jpg Primers are used to amplify areas with VNTRs, which differ in different chromosomes, different individuals
10_30_2_PCR_forensic.jpg Three areas amplified to generate a DNA fingerprint
Basic 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
Melt template, then rapidly cool * some primers will anneal to complementary sequence 53 53
Melt template, then rapidly cool * some primers will anneal to complementary sequence Add DNA polymerase * provide substrate + time to extend
Melt template, then rapidly cool * some primers will anneal to complementary sequence Add DNA polymerase * provide sunstrate + time to extend
These 3 steps constitute 1PCR ‘cycle’, which will be repeated many times (usually 25-30) 1) melt template 2) anneal oligonucleotide primers 3) extend with DNA polymerase If ever confused about how PCR works… * draw out first three cycles 25-30x
Limitations – finite amounts of * dNTPs * primers * DNA pols Exhaustion after 30
Good Primer’s Characteristic A melting temperature (Tm) in the range of 52 C to 65 C Absence of dimerization capability Absence of significant hairpin formation (>3 bp) Lack of secondary priming sites Low specific binding at the 3' end (ie. lower GC content to avoid mispriming)
Uniqueness 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) Primer candidate 1 5’-TGCTAAGTTG-3’ Primer candidate 2 5’- CAGTCAACTGCTAC-3’ TGCTAAGTTG CAGTCAACTGCTAC Template DNA 5’...TCAACTTAGCATGATCGGGTA...GTAGCAGTTGACTGTACAACTCAGCAA...3’ NOT UNIQUE! UNIQUE! TGCT AGTTG A
Length Primer 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.
PANJANG PRIMER Panjang primer 8 4 pangkat 8 = pb Ukuran kromosom kb ada kemungkinan situs Panjang primer 17 = pb diharapkan hanya menempel pada 1 situs
Base Composition Base 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. Usually, 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. Template DNA 5’...TCAACTTAGCATGATCGGGCA...AAGATGCACGGGCCTGTACACAA...3’ GCCCG TGCCCGATCATGCT GCCCG GCCCG CAT T T AT GC
Melting Temperature Melting 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. Calculation Shorter than 13: Tm= (wA+xT) * 2 + (yG+zC) * 4 Longer than 13: Tm= *(yG+zC-16.4) /(wA+xT+yG+zC) (Formulae are from
Annealing Temperature T anneal = T m_primer – 4 C Annealing Temperature, T anneal – the temperature at which primers anneal to the template DNA. It can be calculated from T m.
Primer Pair Matching Primers 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 T anneal are, the better.
Summary ~ when is a “primer” a primer? 5’3’ 5’ 3’
Summary ~ Primer Design Criteria 1.Uniqueness: ensure correct priming site; 2.Length: bases.This range varies; 3.Base composition: average (G+C) content around 50-60%; avoid long (A+T) and (G+C) rich region if possible; 4.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. 5.Melting Tm between C are preferred; 6.Assure that primers at a set have annealing Tm within 2 – 3 C of each other. 7.Minimize internal secondary structure: hairpins and dimmers shall be avoided.
RT-PCR Procedure mRNA was isolated from a tissue cell culture Reverse transcriptase used to synthesize cDNA from mRNA PCR performed on cDNA using Taq polymerase and primers specific for Syk gene Beta-actin used as an external control for the RT- PCR Southern blot used a biotynlated internal Syk probe used to confirm amplification of Syk mRNA
RT-PCR Results Syk is expressed in normal mammary gland tissue and breast epithelial cells Several carcinoma cell lines expressed Syk strongly Some carcinoma cell lines poorly expressed Syk Some carcinoma cell lines completely lack Syk expression
Advantages Disadvantages RT-PCR is much faster and more sensitive that RNase protection assays RT-PCR is very sensitive and can detect low levels of mRNA in cells RT-PCR requires a lot of preparation and must be strictly controlled Non-competitive RT-PCR may result in false conclusions because experimental results are caused by differences in PCR conditions
REAL TIME PCR RT PCR
Apakah 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 bersamaan Dikenal sebagai ‘Rapid Cycle PCR’ dengan siklus temperatur antara detik Produk PCR dapat dianalisa selama proses amplifikasi Menggunakan ‘pewarna DNA’ dan ‘probe fluoresensi’ Data dikoleksi dari tabung yang sama didalam instrumen yang sama Tidak ada transfer sampel, penambahan reagensia atau gel separasi
Apakah Real-Time PCR ? Format deteksi : SYBR Green I Hybridization Probes (HybProbe Probes) Hydrolysis probes / Taqman Probes SimpleProbe Probes Dapat melakukan ‘real-time quantitative PCR’ ‘Real Time PCR’ adalah metode yang ‘powerful’, sederhana dan cepat
Applications For Detecting and Quantifying Transcripts Quantifying viruses Pathogen detection Gene expression Drug therapy DNA damage Immune response Genotyping
Monitoring PCR Reaction Agarose Gel BlottingLightCycler
What’s Wrong With Agarose Gels? l Poor precision. Low sensitivity. Low sensitivity. Short dynamic range < 2 logs. Short dynamic range < 2 logs. Low resolution. Low resolution. Non-automated. Non-automated. Size-based discrimination only. Size-based discrimination only. Results are not expressed as numbers. Results are not expressed as numbers. Ethidium bromide staining is not very quantitative. Ethidium bromide staining is not very quantitative.
Approaches in quantifying by PCR.. 3. Real-time RT-PCR (QPCR). Measurement occurs during exponential phase.
Available 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
1. SYBR Green I Ketika SYBR Green I berikatan dengan dsDNA, akan terjadi peningkatan fluoresensi Selama tahapan PCR yang berbeda, intensitas dari sinyal fluoresensi akan berbeda, tergantung dari jumlah dsDNA yang ada
SYBR Green I
Fluoresces when bound to dsDNA
2. HybProbe Probes Hybridization probe merubah fluoresensi pada saat hibridisasi dengan ‘fluorescene resonance energy transfer (FRET)’
Fluorescence Resonance Energy Transfer (FRET) E E E E
FRET is Nonradiative energy transfer
Polymerization 5’ 3’ 5’ 3’ 5’ Forward Primer Reverse Primer TaqMan ® Probe R Q 3. Taqman probes are “hydrolysis” probes
Displacement 5’ 3’ 5’ 3’ 5’ Forward Primer Reverse Primer R Q
Hydrolysis 5’ 3’ 5’ 3’ 5’ Q R
Polymerization Completed 5’ 3’ 5’ 3’ 5’ R Q
4. Simple Probes Simple Probe adalah bentuk sederhana dari hybridization probe dan hanya menggunakan 1 probe saja Ketika terjadi hibridisasi akan memancarkan sinyal fluoresensi yang lebih besar Perubahan sinyal fluoresensi tergantung dari status hibridasi dari probe, semakin stabil hibridisasinya semakin tinggi temperatur melting Untuk aplikasi SNP genotyping dan deteksi mutasi
Molecular Beacons are “hybridization” probes
Masalah PCR Hasil PCR tidak ada atau sedikit Terlalu banyak pita Pita-pita tidak jelas
Suhu hibridisasi primer Hasil PCR sedikit atau tidak ada Turunkan suhu hibridisas Muncul terlalu banyak pita Naikan suhu hibridisasi
Masalah preparasi DNA Larutan DNA berwarna (kontaminasi) Tidak ada DNA Rasio A260/A280 rendah (gula, fenol, protein)