Lecture 18. PCR Technology. Growing PCR Industry

Transcription

1 Lecture 18 PCR Technology Growing PCR Industry Basic PCR, Cloning of PCR product, RT-PCR, RACE, Quantitative PCR, Multiplex PCR, Hot start PCR, Touchdown PCR,PCR sequencing.. How PCR started The DNA duplex would be denatured to form single stands. This denaturation step would be carried out in the presence of a sufficiently large excess of the two appropriate primers. DNA polymerase will be added to complete the process of repair The whole cycle could be repeated.(h.g. Khorana, 1971, JMB 56, 341) I stopped the car again and started drawing lines of DNA molecules hybridizing and extending, the product of one cycle becoming the templates for the next in a chain reaction (K.B. Mullis, 1990, Sci Am 262, 56

2 The Nobel Prize in Physiology or Medicine 1968 The Nobel Prize in Physiology or Medicine 1975 "for their interpretation of the genetic code and its function in protein synthesis" "for their discoveries concerning the interaction between tumour viruses and the genetic material of the cell" Robert W. Holley Har Gobind Khorana Marshall W. Nirenberg 1/3 of the prize 1/3 of the prize USA USA USA Cornell University Ithaca, NY, USA b d University of Wisconsin Madison, WI, USA b (in Raipur, India) 1/3 of the prize National Institutes of Health Bethesda, MD, USA b David Baltimore 1/3 of the prize Renato Dulbecco 1/3 of the prize USA USA USA Massachusetts Institute of Technology (MIT) Cambridge, MA, USA Imperial Cancer Research Fund Laboratory London, United Kingdom Howard Martin Temin 1/3 of the prize University of Wisconsin Madison, WI, USA b b (in Catanzaro, Italy) b d. 1994

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4 First three PCR cycles

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7 Basic Polymerase Chain Reaction A thermostable DNA polymerase to catalyze template-dependent synthesis A pair of synthetic oligonucleotides to prime DNA synthesis Deoxynucleoside triphosphates (dntps) Divalent cations Buffer to maintain ph Monovalent cations Template DNA Template DNA (10 5 to 10 6 molecules) 20 pmol of each primer 20 mm Tris-HCl (ph 8.3 at 20 C) 1.5 mm MgCl2 25 mm KCl 0.05% Tween µg/ml autoclaved gelatin or nuclease free BSA 50 µm dntp each 2 units of Taq DNA polymerase Total volume 100 µl

8 Basic Polymerase Chain Reaction (92-98 C) Denaturation 96 C for 15 sec (37-70 C) Primer annealing 55 C for 30 sec (70-74 C) Primer extension 72 C for 90 sec Repeat for 20 to 30 cycles (70-74 C) Final extension 72 C for 5 min Stop the reaction: Chill at 4 C or adding EDTA to 10 mm PCR products gel purified for further work Length: 18 to 28 nt Primer Design GC content: 50 to 60% Tm: 55 to 80 C Others: avoid 3 -end complementarity Anneal Temperature = 2 x ( A + T ) + 4 x ( G + C )

9 To clone blunt-ended PCR fragments amplified with Platinum Pfx, which can amplify genomic templates for up to 12 kb and plasmid template up to 20 kb. See The single 3 adenosyl extension generated by Taq DNA polymerase provides a highly efficient method to clone PCR products into a vector (T vector) containing complementary unpaired 3 thymidyl residue. T vector can be generated by (I) digesting the vector with XcmI, MboII, or HphI; (ii) using terminal transferase to add dideoxyttp to the 3 terminai of a linearized vector; (iii) using the template-independent terminal transferase activity of Taq DNA pol to add a T to the terminal 3 -OH of a linearized vector

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12 RT-PCR Reverse transcriptase-pcr is to amplify cdna copies of RNA It is used to retrieve and clone the 5 and 3 termini of mrna It is used to generate large cdna libraries from very small amounts of mrna It can be adapted to identify mutations and polymorphisms It can be used to measure the strength of gene expression 1. Mesophilic enzymes encoded by avian myeloblastosis virus (AMV) or Moloney strain of murine leukemia virus (Mo-MLV). 2. Variants of Mo-MLV reverse transcriptase that lacks RNase H activity. 3, Thermostable Tth DNA polymerase (exhibit reverse transcriptase in the presence of Mn2+ Three ways to amplify cdna Gene specific priming Oligo(dT) proming Random hexamer priming

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14 Control TIME(hour) µM Control 100µM GAPDH 50µM COX-2 Normal cells c-fos GAPDH c-myc COX-2 TK Tumor cells GAPDH PCNA Control BRCA-1 0 COX-2 BRCA-2 CacO2 COX-1 GAPDH COX µM

15 invitrogen tavi/guide.html

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17 PCR-based restriction fragment length polymorphism (PCR-RFLP) includes

18 Inverse PCR Inverse PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available It is frequently used in chromosome walking Inverse PCR is used to clone sequences flanking a known sequence. Flanking sequences are digested and ligated to make a circular DNA. PCR primers pointing away from the known sequences are used to amplify the flanking sequences. For Inverse PCR primer design, please visit Primo Inverse.

19 Rapid amplification of cdna ends

20 Synthesis of 5 and 3 fragments PCR cycles 94 C, 4 min 94 C, 30 sec 30 cycles 60 or 52 C, 1 min 72 C, 1 min 72 C, 10 min * Annealing temperature: 52 C for 5 fragment; 60 C for 3 fragment. * Primer concentration is 0.1 µm in all PCR. marker 5 fragment (60 C) 3 fragment (60 C) 5 fragment (60 C) 5 fragment (52 C) marker Full length (52 C) Template, ComA, and A (no PCR) Template only (1 µl) 0.9% agarose Purification of 5 and 3 fragments The gel slices were dissolved in the Binding buffer of High Pure PCR Product Purification Kit (Roche) by incubated at 55 C for 10 min. The rest procedure was according to the company s instruction. The purified fragments were dissolved in 100 µl TE, from which 5 µl was used in the following PCR to synthesize the full-length, mutagenized HSF1.

21 Hot start PCR Top: in conventional PCR, mispriming events that occur on the initial upramp can lead to amplification of non-specific products. Bottom: in hot start PCR, active polymerase is present only at temperatures above the specific annealing temperature. The 10-min preincubation serves to fully denature any mispriming and to initiate activation of enzyme (AmpliTaq Gold enzyme from PE Applied Biosystem

22 Real Time PCR : Ability to measure the concentrations of nucleic acids over a vast dynamic range; its high sensitivity; its capacity to process many samples simultaneously.