Site directed mutagenesis by pcr

This describes about the basic principle behind the site directed mutagenesis by pcr............

1. Site Directed Mutagenesis By PCR Submitted By , Submitted To , POORANACHITHRA M DR.P.S . SUDHAKAR GANDHI, Ist M.Th. Biotechnology, Assistant Professor, Dept. Of Biotechnology Dept . Of Biotechnology, Bharathidasan Institute Of Technology, Bharathidasan Institute Of Technology, Anna University, Trichy-620024. Anna University, Trichy-620024

2. Site Directed Mutagenesis Site-directed mutagenesis is a  molecular biology  method that is used to make specific and intentional changes to the  DNA sequence  of a  gene  and any  gene products. Also called as site-specific mutagenesis  or oligonucleotide-directed mutagenesis. This can be done by using oligonucleotides in a primer extension method with DNA polymerase (in PCR), developed by   Michael Smith  who was awarded a Nobel Prize in 1993 for this contribution.

3. Principle Behind PCR Based SDM The principle of site-directed mutagenesis is that a mismatched oligonucleotide is extended, incorporating the "mutation" into a strand of DNA that can be cloned. Here the synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so it can  hybridize with the DNA in the gene of interest The single-strand primer is then extended using a DNA polymerase, which copies the rest of the gene .

4. Different Approaches Of PCR based SDM Site-directed is typically performed using PCR in 2different ways, 1.normal PCR with mutated primer 2. primer extension method Primers designed with mutations can introduce small sequence changes primer extension can be used to achieve longer mutant regions .

5. Existing sequence 5′ggacgcaagc-------------aggacattga 3′ 3′cctgcgttcgac------------- tcctgtaact 5’ Desired sequence 5′gga TC caagc-------------aggacattga 3′ 3′cct AG gttcgac------------- tcctgtaact 5 ′ PCR for Base Substitutions.

6. PCR for Terminal Additions PCR for Deletions

7. Primer Extension for an Insertion: Primers B and C contain the complementary sequence that will be inserted (indicated by the blue line). The first round of PCR uses two reactions with primer pairs A/B (1) and C/D (2). The two resulting PCR products are mixed together with primer pair A/D for a second round of PCR.

8. The overlapping regions of the two, first-round PCR products allow the strands to hybridize and the second round of PCR creates the final, fulllength product with the desired insertion.

9. Primer Extension for Délétions: Primers B and C are located on either side of the sequence to be deleted and contain sequence from both sides of the deletion (indicated by black or gray additions that match the black or gray original sequence ). This sequence will allow them to overlap with the other fragment after the first round of PCR. The first round of PCR uses primer pairs A/B and C/D.

10. The two resulting PCR products are mixed together with the primer pair A/D for a second round of PCR. The overlapping regions of these two, first-round PCR products allow the strands to hybridize and the second round of PCR creates the final, full-length product with the desired area deleted.

11. Primer Extension for Longer Additions.

12. Applications Site-directed mutagenesis is used to generate mutations that may produce rationally designed protein that has improved or special properties (i.e. Protein engineering) This method of altering the sequence allows researchers to investigate the impact of sequence changes, such as single nucleotide polymorphisms (SNPs ), or to insert or delete a sequence element, such as a ligand binding site or restriction site .

13. With PCR based methods it is hard to replicate the mutated DNA, in order for replication to occur super competent cells must be used and are expensive! Screening can be tedious, usually requires sequencing to confirm if mutation occurred . Limitations

14. Reference Site-directed mutagenesis - From Wikipedia, the free encyclopedia PCR-Based Site-Directed Mutagenesis - Atsushi Shimada Site-directed mutagenesis - Albert Jeltsch , Thomas Lanio A simple method for site-directed mutagenesis using the polymerase chain reaction - Anne Hemsley, Norman Arnheim, Michael Dennis Toney, Gino Cortopassi and David J.Galas Ultramer ™ Oligonucleotides - Adam Clore PhD , Brian Reinertson, and Scott Rose PhD

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