Journal Club & MSc Seminar

Transcription

1 Journal Club & MSc Seminar

2 2

3 The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention A special DNA polymerase (Taq) is used to make many copies of a short length of DNA (100-10,000 bp) defined by primers Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry 3

4 4

5 Development. PCR work was first published (1985)using Klenow polymerase unstable with heat New enzyme had to be added manually at each step Maximum length 400bp Great idea not very practical First reports using DNA polymerase from Thermus aquaticus (1988) Taq-polymerase (Saiki et al, 1988) from Yellow stone National Park hot springs Developed automatic thermocycler programmable heat block 5

6 6

7 Polymerase Chain Reaction (PCR) PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested. The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur. 7

8 PCR Thermocycler 8

9 What all PCR Can Do? Starting with one original copy an almost infinite number of copies can be made using PCR Amplified fragments of DNA can be sequenced, cloned, probed or sized using electrophoresis Defective genes can be amplified to diagnose any number of illnesses Genes from pathogens can be amplified to identify them (i.e., HIV, Vibrio sp., Salmonella sp. etc.) Amplified fragments can act as genetic fingerprints 9

10 PROCEDURE.. 10

11 PCR Reagents 1X Buffer 10mM Tris-HCl, 50mM KCl MgCl 2 1mM - 4mM (1.5mM) dntps 200μM Primers 100nM-1μM, 200nm (or less) for real time analysis DNA polymerase Taq DNA polymerase is thermostable 1-4 Units (1 unit) DNA 10pg-1μg (20ng) 11

12 Different types of buffers 12

13 13

14 Polymerase Chain Reaction 14

15 15

16 Initiation - Forming the Replication Eye Origin of Replication 16

17 Extension - The Replication Fork Laging Strand Okazaki fragment DNA Polymerase RNA Primers Primase Single strand binding proteins Helicase Leading Strand 17

18 How are the functions of replication achieved during PCR??? Function Melting DNA Polymerizing DNA Providing primer Joining nicks. Heat PCR. Taq Polymerase. Primers added to the reaction mix. N/A as fragments are short ENZYMES Helicase SSB proteins Topoisomerase DNA pol Primase Ligase 18

19 Temperature PCR 100 Melting 94 o C 50 0 T i m e 19

20 Temperature PCR 100 Melting 94 o C 50 0 T i m e Heat 20

21 Temperature PCR Melting 94 o C Annealing Primers 50 o C Extension 72 o C Melting 94 o C 0 T i m e 21

22 Temperature PCR Melting 94 o C Annealing Primers 50 o C Extension 72 o C Melting 94 o C 30x 0 T i m e Heat Heat 22

23 Temperature PCR Melting 94 o C Annealing Primers 50 o C Extension 72 o C Melting 94 o C 30x 0 T i m e 23

24 Temperature PCR Melting 94 o C Annealing Primers 50 o C Extension 72 o C Melting 94 o C 30x 0 T i m e Heat Heat supervised by: Dr. Aslanimehr 24

25 Temperature PCR Melting 94 o C Annealing Primers 50 o C Extension 72 o C Melting 94 o C 30x 0 T i m e supervised by: Dr. Aslanimehr 25

26 Temperature PCR Melting 94 o C Annealing Primers 50 o C Extension 72 o C Melting 94 o C 30x 0 T i m e Fragments of defined length supervised by: Dr. Aslanimehr 26

27 More Cycles = More DNA Size Marker Number of cycles

28 Terrible & Good Results 28

29 PCR Optimisation 1: Buffers Most buffers have only KCl (50mM) and Tris (10mM) Concentrations of these can be altered KCl facilitates primer binding but concentrations higher than 50mM inhibit Taq DMSO, BSA, gelatin, glycerol, Tween-20, Nonidet P- 40, Triton X-100 can be added to aid in the PCR reaction Enhance specificity, but also can be inhibitory Pre-mixed buffers are available 29

30 PCR Optimisation 2: MgCl 2 MgCl 2 : required for primer binding MgCl 2 affects primer binding, Tm of template DNA, productand primer-template associations, product specificity, enzyme activity and fidelity dntps, primers and template chelate and sequester the Mg ion, therefore concentration should be higher than dntps (as these are the most concentrated) Excess magnesium gives non-specific binding Too little magnesium gives reduced yield 30

31 PCR Optimisation 3: Primer Design Specific to sequence of interest Length nucleotides Annealing temperature 50 o C-70 o C Ideally 58 o C-63 o C GC content 40-60% end critical (new strand extends from here) GC clamp (G or C at terminus) Inner self complementarity: Hairpins <5, dimers <9 complementarity: <3-4 bases similar to other primer regions 31

32 PCR Optimisation 4: Cycling Conditions Denaturation: Some Taq polymerases require initial denaturation (hot start) Annealing temperature: ~ 5 o C less than Tm of primers Tm = 4(G + C) + 2(A + T) o C (or use of primer software) Decrease in annealing temperature result in non-specific binding Increase in annealing temperature result in reduced yield 32

33 PCR Optimisation 5: Cycle Number cycles Half-life of Taq is 30 minutes at 95 o C Therefore if you use more than 30 cycles at denaturation times of 1 minute, the Taq will not be very efficient at this point Theoretical yield = 2 n ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc eg. if you start with 100 copies after 30 cycles you will have 107, 374, 182, 400 copies 33

34 In summary Primer length should not exceed 30 mer. Tm, not more than 60 degree. GC Content should be in the range of % for optimum PCR efficiency. Primers should end (3 ) in a G or C, or CG or GC: this prevents breathing of ends and increases efficiency of priming. 34

35 Primer Problems primers should flank the sequence of interest primer sequences should be unique primers that match multiple sequences will give multiple products repeated sequences can be amplified - but only if unique flanking regions can be found where primers can bind 35

36 PCR Based Methods Sequence Specific Oligonucleotide (SSO) probe Amplified fragment-length polymorphism to generate finger prints Large VNTR regions (10-30 b.p. repeat) Short Tandem Repeats (STR) (2-7 b.p. repeat) RAPD using universal primers Rep- PCR (ERIC primers) PCR- Ribotyping (16S rdna regions) 36

37 PCR fingerprinting AP-PCR (arbitrarily primed PCR), 1 primer required, bp, no sequence information required REP-PCR (repetitive extragenic palindromic sequences) 2 primers insert randomly into the REP sites ERIC-PCR (enterobacterial repetitive intergenic consensus sequences), 2 primers insert randomly into the ERIC sites, best for Gram Negative microbes All of these fingerprinting techniques tell one if two isolates are the same or different. They do not provide information about the identity or relatedness of the organisms 37

38 RFLP Fingerprinting Analysis RFLP = restriction fragment length polymorphism RFLP analysis involves cutting DNA into fragments using one or a set of restriction enzymes. For chromosomal DNA the RFLP fragments are separated by gel electrophoresis, transferred to a membrane, and probed with a gene probe. of this fingerprinting technique is that all bands are bright (from chromosomal DNA) because they are detected by a gene probe. AP-PCR, ERIC-PCR, and REP-PCR all have bands of variable brightness and also can have ghost bands. For PCR products a simple fragment pattern can be distinguised immediately on a gel. This is used to confirm the PCR product or to distinguish between different isolates based on restriction cutting of the 16S-rDNA sequence ribotyping. Also developed into a diversity measurement technique called TRFLP. 38

39 AP-PCR Arbitrarily Primed PCR (AP-PCR) or Random Amplified Polymorphic DNA (RAPD) are methods of creating genomic fingerprints from species of which little is known about target sequence to be amplified. 39

40 REP-PCR 40

41 ERIC-PCR Pulsed-field gel electrophoresis resolving XbaI restriction fragments from E. coli isolates.lines 3 7, 9, 10 and 11 13, correspond to isolates presented of by: patients moein yeylagh 5 and beigi, 14, respectively. 41

42 Variations of the PCR Colony PCR Nested PCR Multiplex PCR AFLP PCR Hot Start PCR In Situ PCR Inverse PCR Asymmetric PCR Long PCR Long Accurate PCR Reverse Transcriptase PCR Allele specific PCR Real time PCR 42

43 Types of PCR Long PCR: Used to amplify DNA over the entire length up to 25kb of genomic DNA segments cloned Nested PCR: Involves two consecutive PCR reactions of 25 cycles. The first PCR uses primers external to the sequence of interest. The second PCR uses the product of the first PCR in conjunction with one or more nested primers to amplify the sequence within the region flanked by the initial set of primers. Inverse PCR: Used to amplify DNA of unknown sequence that is adjacent to known DNA sequence. Quantitative PCR: Product amplification write time, which is compared with a standard DNA. Hot start PCR: Used to optimize the yield of the desired amplified product in PCR and simultaneously to suppress nonspecific amplification. 43

44 Colony PCR Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products. Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE autoclaved dh2o in an microfuge tube. Heat the mix in a boiling water bath (90-100C) for 2 minutes Spin sample for 2 minutes high speed in centrifuge. Transfer 20 μl of the supernatant into a new microfuge tube Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction. 44

45 Hot Start PCR This is a technique that reduces non-specific amplification during the initial set up stages of the PCR The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95 C) before adding the polymerase DNA Polymerase- Eubacterial type I DNA polymerase, Pfu These thermophilic DNA polymerases show a very small polymerase activity at room temperature. 45

46 Nested PCR Two pairs (instead of one pair) of PCR primers are used to amplify a fragment. First pair -amplify a fragment similar to a standard PCR. Second pair of primers-nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. Advantage- Very low probability of nonspecific amplification 46

47 47

48 Multiplex PCR Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. E. Coli genome Salmonella sp. genome or 48

49 Inverse PCR Inverse PCR (Ochman et al., 1988) uses standard PCR (polymerase chain reaction)- primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been selfligated Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA. Application Amplification and identification of flanking sequences such as transposable elements, and the identification of genomic inserts. 49

50 Long PCR Extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb). Long PCR is useful only if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together. Application- to clone large genes 50

51 Reverse Transcriptase PCR Based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses. First step of RT-PCR - "first strand reaction -Synthesis of cdna using oligo dt primers (37 C) 1 hr. Second strand reaction -Digestion of cdna:rna hybrid (RNaseH)-Standard PCR with DNA oligo primers. Allows the detection of even rare or low copy mrna sequences by amplifying its complementary DNA. 51

52 Viral RNA Bacterial mrnaprotozoan(eukaryotic) RT-PCR The enzyme reverse transcriptase is used to make a DNA copy (cdna) of an RNA template from a virus or from mrna. RNA Extension Reversetranscriptase Primer RNA/cDNA RNA Normal PCR with two primers cdna 52

53 Why real time PCR? QUANTITATION OF mrna northern blotting ribonuclease protection assay in situ hybridization RT-PCR most sensitive can discriminate closely related mrnas technically simple but difficult to get truly quantitative results using conventional PCR 53

54 Real-Time PCR Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

55 Traditional PCR has advanced from detection at the endpoint of the reaction to detection while the reaction is occurring (Real-Time). Real-time PCR uses a fluorescent reporter signal to measure the amount of amplicon as it is generated. This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles. 55

56 Real-time PCR advantages * Amplification can be monitored real-time * No post-pcr processing of products (high throughput, low contamination risk) * Ultra-rapid cycling (30 minutes to 2 hours) * wider dynamic range of up to fold * requirement of 1000-fold less RNA than conventional assays (6 picogram = one diploid genome equivalent) * detection is capable down to a two-fold change * confirmation of specific amplification by melting curve analysis * most specific, sensitive and reproducible * not much more expensive than conventional PCR (except equipment cost)

57 Real-time PCR disadvantages * Not ideal for multiplexing * setting up requires high technical skill and support * high equipment cost * intra- and inter-assay variation * RNA liability * DNA contamination (in mrna analysis)

58 Applications of PCR Classification of organisms Genotyping Molecular archaeology Mutagenesis Mutation detection Sequencing Cancer research Detection of pathogens DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis 58

59 PCR Virtues High sensitivity Can detect and quantify specific events Higher stability of DNA permits analysis of food samples. Quantitative and qualitative 59

60 Thank you THANK YOU 60