PCR AND ITS VARIANTS

IN BRIEF ABOUT POLYMERASE CHAIN REACTION.

1. PCR AND ITS VARIANTS BY : SHILPA C ( PALB9313) SOWMYAPRIYA R (PALB9315)

2. Polymerase chain reaction (PCR) is a technique that results in exponential amplification of a desired region of a DNA molecule in vitro using a set of the sequence-specific complementary primers in the enzymatic cyclic temperature dependent reaction. 2

3. The of PCR technique was invented by Kary Mullis , a Research Scientist at a California Biotech Company, in 1983 . For this work, Mullis received the 1993 Noble Prize in Chemistry. Working principle: As the name implies , it is a chain reaction ,a small fragment of the DNA of interest which serves as the template for producing the primers that initiate the reaction. One DNA molecule is used to produce 2 copies, then 4 copies ,then 8 copies and so forth.. 3

4. COMPONENTS OF PCR 4

5. Steps in PCR : 5

6. PCR Steps Initial Denaturation Denaturation Annealing Extension Final extension Temperature 90 ̊C-95 ̊C 90 ̊C-95 ̊C 55 ̊C-6o ̊C 72 ̊C 72 ̊C Time 5min 1min 50sec 1min 7 min ——————– ——————- 25-28 cycles ————— ——————— The reaction conditions are, 6

7. Why different types of PCR ? Reduce contamination caused due to non-specific binding ( Primer dimer, misprimed or false primed targets) leads to wrong amplification. If unknown flanking DNA sequence adjacent to gene of interest If RNA is there Whether amplification is taking place or not What is the amplified quantity. More than one template as to be amplified at the same time. One side sequence is known to design only one primer Detect mutation 7

8. VARIANTS OF PCR 8

9. ANCHORED PCR W here only enough information to make a single primer is known , for second primer a known sequence is added to the end of the DNA by enzymatic addition of a polynucleotide stretch of homopolymer tailing of cDNA catalyzed by the terminal deoxynucleotidyl transferase or by ligation of known sequence. This technique of amplification with single sided specificity has been known as one-sided PCR or anchored PCR . 9

10. Applications •The production of cDNA libraries from very small amounts of starting material Limitations It is relatively difficult protocol. Large amount of starting templates are required . 10

11. NESTED PCR Designed to increase the sensitivity and specificity. It involves the use of two primer sets directed against the same target and two successive PCR reactions . The first set of primers is designed to anneal to sequences upstream from the second set of primers, whereas the second set of primers is situated internally or nested with respect to the first set of primers. First set of primers also called  “outer primers”  amplify a large fragment of the gene which is used as a template in the second round of PCR that targets a smaller region of the amplicon using the second set of primers also known as  “inner primers or nested primers.” 11

12. Application Nested PCRs have proven valuable for the detection of microorganisms when they are present in very low quantities. Limitations Susceptible to contamination:  The extreme sensitivity of nested PCR comes with its own set of problems. Contamination mostly occurs during the transfer of the first-round product to the second tube for the second round of amplification. Costly:  This PCR assay is also more costly as it involves the use of two separate reactions to arrive at one result. 12

13. LIGATION MEDIATED PCR (LM-PCR) Ligation-mediated PCR uses small DNA oligonucleotide linkers or adaptors that are first ligated to fragments of the target DNA . PCR primers that anneal to the linker sequences are then used to amplify the target fragments. 13

14. USES OF LM PCR : Adapted to map DNA damage and reveal DNA–protein interactions inside living cells . Determination of primary nucleotide sequences. Determination of cytosine methylation pattern. DNA sequencing, genome walking, and DNA footprinting . 14

15. MULTIPLEX PCR Amplifying different DNA sequences or different DNA templets by using the different set of primers in a single PCR reaction is refers to as a multiplex PCR . Based on the type of the template the multiplex PCR can be divided into two categories: Single template PCR reaction : This technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template 2. Multiple template PCR reaction : T his technique uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primer may lead to cross hybridization with each other and the possibility of mis -priming with other templates. 15

16. Uni -template multiplex PCR is used in the detection of inherited genetic disorders. M ulti-template multiplex PCR is not a valid technique for the detection of inherited genetic disorders . It is applicable in the detection of different strains or species of pathogens. 16

17. How to Avoid Primer Dimer Formation ? Annealing temperatures for each of the primer sets must be optimized. Base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis. Limitations of multiplex PCR : Although the technique is advantageous, the multiplexing is not applicable to all types of reaction. (For the larger amplicon such as 800bp or 1000bp, multiplex PCR might not work efficiently always) 17

18. Applications : 18 Multiplex PCR in combination with the real-time PCR is even more valuable and useful in the quantitative studies.

19. HOT START PCR Which reduces the non-specific bindings by limiting one of the reagents until the heating step of the PCR. Hot start PCR = One of the components  starts  its activity under the  hot  condition of  PCR . The aim of the Hot start PCR is to limit the reaction at an early stage, by limiting Taq DNA polymerase in the reaction. 19

20. Advantages: The hot start PCR technique decreases the nonspecific bindings. Also, it prevents mis -priming and primer dimer formation. By using the hot start Taq DNA polymerase, the reaction can even be prepared at room temperature. It increases the yield and accuracy of the results. Disadvantages: The overall cost of the reaction is increased, due to the use of the antibody. The heating step is predominant in the hot start PCR, hence due to the higher temperature for a longer time the template DNA can damage or break down badly. 20

21. TOUCHDOWN PCR “By sequentially decreasing the annealing temperature during each PCR cycle, the chance of the non-specific binding can be reduced .” B y using higher annealing temperatures at the earlier cycles, only very specific base pairing between the primer and the template will occur I ncreases the efficiency by lowering the annealing temperatures gradually toward the end of cycles. 21

22. The annealing temperature   of the primers is the temperature at which the primers bind to its specific complementary sequence on DNA. The melting temperature  is a temperature at which the primer dissociate from the complementary DNA sequence . T he annealing temperature is 5°C lower than the melting temperature 22

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24. Advantages The touchdown PCR reduces the primer-dimer formation capacity of primers. It will also provide higher specificity by reducing the non-specific and unwanted bindings of the primer to the template DNA. The technique is extremely useful in the templates having higher GC contents.   Disadvantages Although it is highly specific, if not performed well the touchdown PCR also gives non-specific results . 24

25. COLD(Co-amplification at lower denaturation temperature) PCR Common PCR will amplify both the major ( wildtype ) and minor (mutant) alleles with the same efficiency , the ability to easily detect the presence of low-level mutations.  COLD –PCR has the ability to preferentially amplify and identify minority alleles and low-level DNA mutations in the presence of excess wildtype alleles is useful for the detection of mutations. 25

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27. Full COLD-PCR Denaturation stage. DNA is denatured at a high temperature – usually 94 °C . Intermediate annealing stage. A llows hybridization of mutant and wild type allele DNA to one another. Because the mutant allele DNA forms the minority of DNA in the mixture they will be more likely to form mismatch heteroduplex DNA with the wild type DNA. Melting stage. These heteroduplexes will more readily melt at lower temperatures. Hence they are selectively denatured at the Tc. Primer annealing stage. The homo-duplex DNA will preferentially remain double stranded and not be available for primer annealing. Extension stage. The DNA polymerase will extend complementary to the template DNA. Since the heteroduplex DNA is used as template, a larger proportion of minor variant DNA will be amplified and be available for subsequent rounds of PCR. Fast COLD-PCR Denaturation stage and I ntermediate annealing stages are skipped. This is because, in some cases, the preferential amplification of the mutant DNA is so great that ensuring the formation of the mutant/ wildtype heteroduplex DNA is not needed. Thus the denaturation can occur at the Tc, proceed to primer annealing, and then polymerase-mediated extension. Each round of amplification will include these three stages in that order. By utilizing the lower denaturation temperature, the reaction will discriminate towards the products with the lower Tm – i.e. the variant alleles. 27

28. Applications : Useful for the detection of mutations 28

29. Advantages: Single-step method capable of enriching both known and unknown minority alleles irrespective of mutation type and position . Does not require any extra reagents or specialized machinery. Therefore, the cost is not increased. Better than conventional PCR for the detection of mutations in a mixed sample. Disadvantages : A suitable critical temperature may not be available that differentiates between mutant and wildtype DNA sequences. Restricted to analyzing sequences smaller than approximately 200bp. No guarantee that all low-level mutations will be preferentially enriched. 29

30. INVERSE PCR It is a variant of PCR that is used to amplify DNA with only one known sequence . limitation of conventional PCR is that it requires primers complementary to both termini of the target DNA, It is commonly used to identify the flanking sequences around genomic inserts. The inverse PCR method includes a series of digestions and self-ligations with the DNA being cut by a restriction endonuclease. This cut results in a known sequence at either end of unknown sequences. 30

31. Primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle . Target DNA - restriction endonuclease digestion . • Self-ligation - gives a circular DNA ligation product. • Target DNA is then restriction digested with a known endonuclease . This generates a cut within the known internal sequence generating a linear product with known terminal sequences. • Standard PCR is conducted with primers complementary to the now known internal sequences. 31

32. SIGNIFICANCE : Identification of unknown flanking regions. For example, the identification and investigation of promoter and enhancer regions of DNA upstream or downstream to the exon region can be possible by using the inverse PCR . In the site-directed mutagenesis . Construction of end specific probes for chromosome walking . It is further useful in the identification of unknown mutations such as gene rearrangements, gene fusion, oncogenic gene arrangement on a chromosome . The inverse PCR is the first choice for transposable element studies, identification and characterization. 32

33. RT (Reverse Transcriptase) PCR It is used for amplifying DNA from RNA . Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. The RT-PCR is broadly divided into two steps, reverse transcription amplification . Depending upon that the RT- PCR can be performed by two methods: One-step RT-PCR Two-step RT-PCR 33

34. Both the reverse transcription and the amplification can be performed in the single reaction. Contamination is very less in comparison with two-step RT-PCR. The major limitation of the one-step RT-PCR : 1.the stock of cDNA 2.non-specific bindings and  primer dimers formation  are also higher in this method because one-step RT PCR utilizes only sequence-specific primers. First reaction, the cDNA is synthesised from the RNA for that reverse transcriptase In the second reaction, instead of reverse transcriptase, the normal DNA polymerase along with other PCR essentials are added into the tube . 34

35. Applications : RT-PCR is widely used in expression profiling , to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. Scientists are working on ways to use RT-PCR in cancer detection to help improve and monitor response to theraphy . 35

36. REAL TIME PCR / Quantitative PCR A real-time polymerase chain reaction (real-time PCR), also known as  quantitative polymerase chain reaction (qPCR) key feature - amplification of DNA is detected in real time as PCR is in progress by the use of fluorescent reporter. The fluorescent reporter signal strength is directly proportional to the number of amplified DNA. Based on the molecule used for the detection Non-specific Detection using DNA Binding Dyes - that  intercalate  with any double-stranded DNA Specific Detection Target Specific Probes - consisting of  oligonucleotides  that are labelled with a  fluorescent  reporter 36

37. RT-PCR can be divided into four stages: Linear ground phase - PCR is just starting, fluorescent signal has not risen above background. Early exponential phase - fluorescent signal just rise significantly above background, the cycle at which occurs is called cycle threshold (Ct). Linear exponential phase (log phase) - PCR is in its optimal amplification stage with doubling PCR products in every cycle. Plateau phase - substrates are exhausted and Taq DNA polymerase is in its end of life, fluorescent signal will no long increase 37

38. Non-specific Detection using DNA Binding Dyes - that  intercalate  with any double-stranded DNA PRINCIPLE : Nonspecific fluorescent dye SYBR Green bind to double-stranded DNA , their fluorescence increases by 20–100-fold. As the amount of double-stranded DNA increases during PCR process, the SYBR f luorescent signal increases correspondingly . LIMITATION SYBR can bind any double-stranded DNA, even  primer dimers  . So it is critical to optimize PCR reactions to amplify the target  amplicon  only. 38

39. Specific Detection Target Specific Probes - oligonucleotides  that are labelled with a  fluorescent  reporter, which permits detection only after  hybridization  of the probe with its complementary sequence. TaqMan  with a reporter fluorescent dye at 5′ end and a quencher dye at 3′ end. When the probe is not hydrolyzed by Taq DNA polymerase, reporter dye emitted fluorescent light is absorbed by quencher dye . When probe is hydrolyzed by Taq DNA polymerase, the 5′ reporter dye is separated from quencher dye and thus 5′ reporter dye fluorescent light will be detected by RT-PCR instrument. The released 5′ reporter dye signal is proportional to the amount of PCR products. 39

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41. Real Time PCR VS Traditional PCR Real time PCR  allows for the detection of PCR product during the early phases of the reaction. Traditional methods use gel electrophoresis for the detection of PCR amplification in the final phase or at end-point of the PCR reaction. APPLICATIONS : Determination /monitoring of viral load Quantification of cancer genes Microarray verification Transgenic copy 41

42. COLONY PCR Colony PCR is a rapid, high throughput PCR method to determine the presence or absence of the inserted DNA into plasmid directly from the bacterial colonies . Colony PCR is a novel method in which by designing the inserted DNA specific primers, we can identify whether our DNA of interest is inserted into the plasmid or not. Colony PCR is the modification of the conventional PCR in which the bacterial colonies are directly used as a PCR template. 42

43. Primers used in Colony PCR : The bacterial colony containing the plasmid can directly be amplified using two or three sets of primers . Insert specific primers  bind to the specific location on both the side of the inserted DNA of our interest which amplify the insertion sequence . vector-specific flanking primers , which amplifies the plasmid DNA other than the inserted DNA (flanking regions on both the side of insert). It helps to determine the size of the insert . It expands regions other than the insert DNA. Orientation-specific primers   are unique primers in which one primer binds inside the insert and another primer binds to the plasmid DNA sequence (sequence other than the insert DNA). This types of primer set provide information about the orientation of inserted DNA of our interest . 43

44. Advantages of colony PCR : Identification of correct ligation and insertion of insert DNA into bacteria as well as yeast plasmid. Colony PCR is a fast and reliable method for the screening of recombinants . This method can easily be used for cDNA library screening Disadvantages of colony PCR: Any mutation in the insert cannot be detected. T he sequence information can not be obtained by the colony PCR. we need to do sequencing for the confirmation of the DNA transformation The chance of false-positive results is high . Use only a few colonies, as many colonies increase the chance of the non-specific bindings. As an insert use short DNA sequences, longer DNA sequences increase the chance of non-specific bindings and PCR reaction failure. 44

45. ASYMMETRIC PCR T he reaction amplifies one DNA strand in a double-stranded DNA template. Thus it is useful when amplification of only one of the two complementary strands is needed such as in sequencing and hybridization probing . The amount of primer for the targeted strand is much more than that of the non-targeted strand. 45

46. Limitation : Asymmetric PCR is not widely used because it has low reaction efficiency and it is hard to optimize the proper primer ratios, the amounts of starting material, and the number of amplification cycles. Limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature . Recently , this process has been changed to be known as Linear-After-The –Exponential-PCR (LATE-PCR) where the limiting lower concentration primer has a higher melting temperature than the higher concentration primer to maintain reaction efficiency.   46

47. SITE DIRECTED MUTAGENESIS using PCR Introduce specific and intentional mutations to the DNA sequence of a gene or any genetic products Short DNA primer contains the expected mutation and is complementary to the template DNA so it can hybridize with the DNA sequence of interest. Thus the amplified gene then contains the mutated site, which are then incorporated into a host cell as a vector and cloned. APPLICATION : Study the function of a gene or protein, or for creating variants of an enzyme with new and improved functions 47

48. METHYLATION SPECIFIC PCR It is one of the most commonly used methods for gene/sequence- specific  detection of DNA  methylation . The DNA undergoes bisulfite conversion of cytosine to uracil and then the  methylated  sequences are selectively amplified with primers  specific  for  methylation . 48

49. Treat sample DNA with sodium bisulfite Sodium bisulfite treatment converts unmethylated cytosine to uracil PCR primers are designed for methylated and unmethylated DNA Final products of unmethylated DNA will contain Ts in place of methylated 49

50. Reasons Variant Reduce contamination caused due to non-specific binding (Primer dimer , misprimed or false primed targets) leads to wrong amplification. If unknown flanking DNA sequence adjacent to gene of interest If RNA is there Amplifies one DNA strand in a double-stranded DNA What is the amplified quantity with change in time Whether amplification is taking place or not More than one template as to be amplified at the same time. One side sequence is known to design only one primer Detect mutation Introduce mutation 50 NESTED PCR HOT START PCR TOUCHDOWN PCR LIGATION MEDIATED PCR INVERSE PCR REVERSE TRANSCRIPTASE PCR ASYMMETRIC PCR REAL TIME PCR MULTIPLEX PCR ANCHORED PCR COLD PCR SITE DIRECTED MUTAGENESIS USING PCR

51. PROBLEMS AND SOME SIMPLE SOLUTIONS No PCR product Check the template DNA concentrations and dilute them if necessary. Try to lower the annealing temperature in the PCR cycle and run 5-10 cycles at very low annealing and extension temperatures (42°C and 68°C for instance), then run another 30-40 cycles at higher temperatures. Check the primer concentrations; if there are faint bands. Bright bands in the well of the gel Caused by over amplification of the PCR product. For this, first dilute the template DNA and then raise the annealing temperature (>55°C). 51

52. Smearing of PCR products or multiple bands Use a low concentration of template DNA and increase the annealing temperature by 2-5°C. Standardize the optimum magnesium concentrations by amplifying at varying concentrations. Then, decrease the number of cycles. Bands in the blank Check all the solutions carefully and change the solutions if there is any uncertainty. Wash the pipettes thoroughly and check for any contamination. 52

53. 53 PCR in agriculture Product development include gene discovery , transformant identification, screening and characterization Seed quality control . Detection of pathogen species-specific identification multiplexing, allowing for identification of more than one species in a single reaction. Grain handling and grain processing industry Unapproved events- To verify the presence or absence of genetically modified(GM) material during importing /exporting . GM content - to quantify the amount of GM present in product(grain) Non-GM crop labeling For presence of high value commodity –GM crop that conveys the characteristic like low phytate maize ,soybean with altered oil profile. Nutrigenomics – linking genetic information to information about food that might be better /worse for a particular condition , in detection of food allergens F or protecting against adulteration and ensuring standardization of processed foods and herbal medicine.

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