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In vitro site-directed mutagenesis is an invaluable technique for studying protein structure-function relationships, gene expression and vector modification. Several methods have appeared in the literature, but many of these methods require single-stranded DNA as the template. The reason for this, historically, has been the need for separating the complementary strands to prevent reannealing. Use of PCR in site-directed mutagenesis accomplishes strand separation by using a denaturing step to separate the complementing strands and allowing efficient polymerization of the PCR primers. PCR site-directed methods thus allow site-specific mutations to be incorporated in virtually any double-stranded plasmid; eliminating the need for M13-based vectors or single-stranded rescue. Several points should be mentioned concerning site-directed mutagenesis using PCR. First, it is often desirable to reduce the number of cycles during PCR when performing PCR-based site-directed mutagenesis to prevent clonal expansion of any (undesired) second-site mutations. Limited cycling which would result in reduced product yield, is offset by increasing the starting template concentration. Second, a selection must be used to reduce the number of parental molecules coming through the reaction. Third, in order to use a single PCR primer set, it is desirable to optimize the long PCR method. And fourth, because of the extendase activity of some thermostable polymerases it is often necessary to incorporate an end-polishing step into the procedure prior to end-to-end ligation of the PCR-generated product containing the incorporated mutations in one or both PCR primers. A protocol is provided as a facile method for site-directed mutagenesis and accomplishes the above desired features by the incorporation of the following steps: (i) increasing template concentration approximately 1000-fold over conventional PCR conditions; (ii) reducing the number of cycles from 25-30 to 5-10; (iii) adding the restriction endonuclease DpnI (recognition target sequence: 5-Gm6ATC-3, where the A residue is methylated) to select against parental DNA (note: DNA isolated from almost all common strains of E. coli is Dam-methylated at the sequence 5-GATC-3); (iv) using Taq Extender in the PCR mix for increased reliability for PCR to 10 kb; (v) using Pfu DNA polymerase to polish the ends of the PCR product, and (vi) efficient intramolecular ligation in the presence of T4 DNA ligase.E importante che gli oligonucleotidi usati come primers e che contengono la mutazione siano lunghi a sufficienza per permettere l'appaiamento anche se la complementarietà non è totale

Mutagenesi con PCR

La reazione di mutagenesi con PCR avviene in due fasi e richiede 4 diversi primer e tre diverse reazioni di PCR.

Primer 2 e primer 3 sono disegnati in modo da essere sovrapposti e introdurre la mutazione nei due filamenti di nuova sintesi.

Primer 1 e primer 4 ciascuno complementare ad una delle estremità della regione da mutagenizzare.

Allestire 2 reazioni di PCR distinte:

1) PCR con primer 1 e 2, per amplificare l’estremità 5’ del frammento: il prodotto risultante porta la mutazione all’estremità 3’

2) PCR con primer 3 e 4, per amplificare l’estremità 3’ del frammento: il prodotto risultante porta la mutazione all’estremità 5’

I due prodotti di PCR sono mescolati, si effettua una denaturazione seguita da una rinaturazione in modo che i filamenti delle due reazioni di PCR possono ibridare tra loro tramite il sito mutato.

Solo dalle molecole ibride date dall’appaiamento delle estremità 3’, la DNA polimerasi può produrre la versione mutata del frammento originario che potrà essere successivamente amplificato con la coppia di primer 1 e 4.

Efficienza di mutagenesi molto alta

The basic procedure starts with synthesizing a short DNA primer, containing the desired base change. Next this synthetic primer has to hybridize with a single-stranded DNA containing the gene of interest. Third, the single stranded fragment is extended using DNA polymerase, which copies the rest of the gene. Fourth, the obtained double stranded molecule is introduced into a host cell and cloned. Fifth, mutants are selected for. The same result can be accomplished using PCR with oligonucleotide "primers" that contain the desired mutation. As the primers are the ends of newly-synthesized strands, by engineering a mis-match during the first cycle in binding the template DNA strand, a mutation can be introduced. Because PCR employs exponential growth, after a sufficient number of cycles the mutated fragment will be amplified sufficiently to separate from the original, unmutated plasmid by a technique such as gel electrophoresis, and reinstalled in the original context using standard recombinant molecular biology techniques. For plasmid manipulations, this technique has largely been supplanted by a PCR-like technique where a pair of complementary mutagenic primers is used to amplify the entire plasmid. This generates a nicked, circular DNA which can undergo repair by endogenous bacterial machinery. However, this process does not amplify the DNA exponentially, rather, linearly. Yields are complicated by the fact that the product DNA must undergo the nick repair and is not supercoiled, resulting in lowered efficiency of transformation in bacteria. Finally, the product DNA is of the same size as the plasmid. Therefore, the template DNA must be eliminated by enzymatic digestion with a restriction enzyme specific for methylated DNA. The template, which for this technique should be biosynthesized will be digested, but the mutated plasmid is preserved because it was generated in vitro and is therefore unmethylated.