Nested pcr

Presentation on nested pcr . contain types of pcr, protocol of nested pcr, advantages of nested pcr, disadvantages of nested pcr, application of nested pcr ,pictorial representation of pcr.

1. PRESENTATION ON NESTED PCR

2. hello! I am HRITIKA SINHA ROLL NO - 1815054028 BRANCH - BIOTECHNOLOGY SEMESTER – 5 TH 2

3. CONTENTS INTRODUCTION OF PCR TYPES OF PCR NESTED PCR PICTORIAL REPRESENTATION OF PROCESS PROTOCOL FOR NESTED PCR ADVANTAGES OF NESTED PCR DISADVANTAGES OF NESTED PCR APPLICATIONS REFERENCES 3

4. 1. POLYMERASE CHAIN REACTION Let’s start with the first set of slides 4

5. PCR is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of  DNA polymerase  to synthesize new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a  primer  to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies ( amplicons ). 5

6. TYPES OF PCR NESTED PCR INVERSE PCR AFLP-PCR ALLELE SPECIFIC PCR ASSEMBLY PCR ASYMMETRIC PCR HOT START PCR COLONY PCR SINGLE CELL PCR REAL TIME PCR / qPCR 6 2.

7. NESTED PCR It is a modification of  polymerase chain reaction  intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites. 7

8. Used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first PCR, one pair of primers is used to generate DNA products, which will be the target for the second reaction. Using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set, thus increasing specificity. Nested PCR is a simple and easy modification of conventional PCR which actually increases the specificity of any reaction. The nested PCR is the best choice in the  microbial identification  and 16s RNA analysis. What are nested pcr 8

9. SETS OF PRIMERS The outer set of primer: The outer primers are primers that are upstream to the inner set of primers. The outer primers are bind to the outside to the flanking region of out target DNA . In the first round of PCR, It is possible that this primer can bind to the site other than the target site and amplifies it. Multiple DNA bands might be observed and lead to false-positive results. The inner set of primer: Even if the non-specific  DNA  sequences can be amplified in the first round of PCR, that non-specific DNA will not be amplified in the second set of amplification.   The second set of primer is specific to the inner sequence (amplicon of the first round of PCR). It is very unlikely that the inner set of primers binds to other than its specific site because the amplicon from the first round of PCR is the template for the second round of amplification. 9

10. 10 PICTORIAL REPRESENTATION OF THE PROCESS

11. 11 Protocol for the nested PCR: In the year 1993,  Kamolvarin  and co-workers described the method for use of two sets of primers for increasing sensitivity and specificity of the PCR. The nested PCR reaction is complete into two steps, a first round of amplification with the outer forward and reverse primers. The protocol is as described, Component Concentration Quantity Master mix 1X 12µL PCR reaction buffer 1X 5µL Outer Forward primer 10pM 1µL Outer Reverse primer 10pM 1µL Template DNA 30ng 3µL Water 3µL Total ——————————- 25µL

12. 12 PCR Steps Initial Denaturation Denaturation Annealing Extension Final extension Temperature 90 ̊C-95 ̊C 90 ̊C-95 ̊C 55 ̊C-60 ̊C 72 ̊C 72 ̊C Time 3min 1min 50sec 1min 7 min ——————– ——————- 25 cycles ————— ——————— After the reaction preparation, put the PCR as shown into the table below, After the completion of the first round of amplification, take the tubes and prepare the reaction for the second round of amplification. Now add 1µL inner forward primer and 1µL inner reverse primer to the PCR reaction tubes of the first round of amplification. Or We can use another method in which, 3µL of PCR product is taken from the first amplification and use it as a template, prepare the reaction as followed.

13. 13 Component Concentration Quantity Master mix 1X 12µL PCR reaction buffer 1X 5µL Inner Forward primer 10pM 1µL Inner Reverse primer 10pM 1µL The amplicon from the first PCR (as a template DNA) —————————————- 3µL Water 3µL Total —————————————- 25µL PCR Steps Initial Denaturation Denaturation Annealing Extension Final extension Temperature 90 ̊C-95 ̊C 90 ̊C-95 ̊C 55 ̊C-60 ̊C 72 ̊C 72 ̊C Time 3min 1min 50sec 1min 7 min ——————– ——————- 35 cycles ————— ——————— Place it back into the PCR machine. Instead of  25 cycles, set the PCR at 35 cycles. Higher amplification is achieved by increasing the cycles in the second round of PCR. we can amplify more amount of gene of our interest. 

14. ADVANTAGES OF NESTED PCR 14 It is beneficial in studies such as phylogenetic analysis and genetic polymorphism. The main advantage of the present method is that it gives 100% accuracy, specificity and sensitivity. For the impossible templates where the GC content might be high or chance of non-specific banding is higher, nested PCR offers the best results. It is also useful in the amplification of genes with the low abundance. Further, nested PCR is the best choice for carcinoma and viral infection studies.

15. DISADVANTAGES OF NESTED PCR 15 The method is time-consuming. Required more reagents such as an extra set of primer and one extra round of agarose gel electrophoresis. Which means the method is quite costly. The chance of contamination is also higher.

16. APPLICATIONS 16 To improve the sensitivity of the assay, nested PCR has been used in many PCR assays. It is particularly useful for suboptimal nucleic acid samples, such as those extracted from formalin-fixed, paraffin-embedded tissue. Nested PCRs have proven valuable for the detection of microorganisms when they are present in very low quantities. For example: detection of  Rickettsia, Bartonella , and similar organisms in blood ( bacteremia ) and tissues, detection of herpesvirus and enterovirus in the CSF, and detection of  M. tuberculosis  in sputum sample.

17. thanks! 17

18. REFERENCES 18 https://geneticeducation.co.in/what-is-nested-pcr/ https://www.google.com/search?q=PIC+OF+NESTED+PCR https://en.wikipedia.org/wiki/Nested_polymerase_chain_reaction https://microbeonline.com/nested-pcr-principle-applications/