M13 Phage as a Vector for DNA Sequencing, Filamentous Bacteriophages, Cloning Vectors, DNA molecules, Chain Termination Method

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Cloning and Expression Vectors Cloning and Expression Vectors Introduction Cloning Vectors for Recombinant DNA Plasmids as Vectors Three Phases of the Development of Plasmid Cloning Vectors Alpha-Complementation for Screening Using "blue-white"Clonies Nonsense Suppressors to Complement Termination Codons in Vectors E.Coli Plasmid Vectors pBR322 and pBr327 Vectors pUC Vectors pSP64-pSP65 pGEM-3, pGEM-4, pGEM-3Z, pGEM-4Z ΠAN13 from ΠAN7 Yeast Plasmid Vectors Retriever Vectors Agrobacterium Plasmid Vectors Lambda Phage (λ)Vectors Lambda Phage (Lysis and Lysogeny) Genetic Markers for the Selection and Screening of λ Vectors Development of Lambda (λ)Phage Vectors Insertion λ Vectors Replacement λ Vectors M13 Phage as a Vector for DNA Sequencing M13-vectors-of-mp-Series Hosts for M13 Vectors Cosmids as Vectors Phagemids as Vectors Phagemids pUC118 and pUC119 Phagemid pBluesript IIKS

Home >> Biotechnology and Genomics >> Cloning and Expression Vectors >> M13 Phage as a Vector for DNA Sequencing

Your Ad Here	M13 phage as a vector for DNA sequencing The filamentous bacteriophages specific to E. coli include M13, f1 and fd. Each of these phages contains a single-stranded closed circular DNA molecule, 6400 nucleotides in length. When used as cloning vectors, these phages have the unique advantage of producing large quantities of recombinant DNA molecules carrying the sequences of one strand of the foreign DNA. Such single-stranded DNAs are the templates of choice (i) for DNA sequencing by the Sanger's dideoxy chain-termination method; (ii) for generating DNA probes that can be rediolabeled in only one strand and used for hybridization and (iii) for site directed mutagenesis using synthetic oligonucleotides.
M13, f1 and fd may be regarded as identical for purposes of their use as vectors, although only M13 has been mainly used for designing vectors. The infecting single stranded DNA is designated as the + (plus) strand, which is converted into replicative double stranded form (RF), in which - (minus) strand is used as the coding strand and for synthesis of + (plus) strands for multiplication of the phage. The M13 genome consists of ten genes (I-X), all of them essential for infection and multiplication of the phage.
There are two intergenic IG regions (between genes II and IV and between genes III and VIII), containing sequences for regulating gene expression and DNA synthesis, so that these sequences are also essential and not dispensable. Therefore, no sequences can be deleted in designing the vectors. Genome of single stranded M13 phage However, in the 500 nucleotides long intergenic region between genes II and IV, foreign DNA can be inserted (this IG region contains signals for packaging and orientation of phage DNA). Insertion of foreign DNA can affect replication of phage genome, but accumulation of mutations in the adjoining genes III or V compensate for this and allow replication. All M13 vectors contain such mutations.	Your Ad Here
Single stranded DNA is generally a poor substrate for restriction enzymes and for ligases needed for inserting a foreign DNA fragment. Therefore, replicative double stranded form of the phage DNA is isolated from infected cells, purified and used like plasmid DNA. After insertion of foreign DNA, the chimeric RF is reintroduced into bacterial cells, where it undergoes the normal replication cycle thus generating single stranded progeny phage particles containing the + strand of the phage along with one of the two strands of the inserted foreign DNA. The other strand (-) of the phage is never packaged.

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