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Submitted By: Deepika Kritaniya Ph.D(BIOTECHNOLOGY)

Ligation restriction

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Page 1: Ligation restriction

Submitted By:

Deepika Kritaniya


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Restriction enzymes were first discovered in the 1960s.

In 1970 Hamilton smith discovered restriction endonuclease from hamophilus influenza strain.

They occur naturally in bacteria where they protect the bacterium against foreign invading DNA from other organisms (e.g. Viruses or phages)

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Type I One enzyme with different subunits for recognition, cleavage, and methylation. Recognizes and methylates a single sequence but cleaves DNA up to 1000 bp away. (eg. SmaI, BamHI)

Type II Two different enzymes which both recognize the same target sequence, which is symmetrical. The two enzymes either cleave or modify the recognition sequence. (eg. HindII)

Type III One enzyme with two different subunits, one for recognition and modification and one for cleavage. Recognizes and methylates same sequence but cleaves 24–26 bp away. (eg. HaeIII , HindIII)

Type IV recognize modified, typically methylated DNA and are exemplified by McrBC of E.coli. Requires at least 2 recognition sites within a distance of 40 – 2,000

Type V utilize guide RNAs to target specific non-palindromic sequences found in organism. They can cut DNA of variable length. The fexibility and ease of use of these enzymes make promising for future genetic engineering applications.

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Some restriction endonucleases cuts on the two DNA strands, leaving two to four nucleotides of one strand unpaired at each resulting end.

These unpaired strands are referred to as sticky ends because they can base-pair with each other or with complementary sticky ends of other DNA fragments.

Other restriction endonuclease cleave both strands of DNA leaving no unpaired bases on the ends, often called blunt ends.

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In 1967 Gellert Lehman, Richardson discovred DNA ligase.

E. coli and phage T4 encode an enzyme, DNA ligase, which seals single-stranded nicks between adjacent nucleotides in a duplex DNA chain. (Olivera et al.1968, Gumport & Lehman 1971).

The T4 enzyme requires ATP, while the E. coli enzyme requires NAD+. In each case the cofactor is split and forms an enzyme–AMP complex. The complex binds to the nick, which must expose a 5′ phosphate and 3′ OH group, and makes a covalent bond in the phosphodiester chain.

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Natural transformation is a mode of horizontal gene transfer (HGT) in bacteria that contributes to the maintenance and evolution of bacterial genomes.

Since the discovery of natural transformation of Streptococcus pneumoniae competent cells, (Griffith, 1928), and the identification of DNA as the transforming principle by Avery,MacLeod and McCarty in 1944 (Avery et al., 1944).

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More than 90 bacterial species have been

shown to be naturally competent eg; B.

subtilis, S. pneumoniae, and Haemophilus influenza,

Neisseria gonorrhoeae , H. pylori etc. (Lorenz and Wackernagel, 1994, Claverys et al., 2000, Dubnau, 1999, Johnsborg and Håvarstein, 2009, Sørensen et al., 2005, Stewart and Carlson, 1986).

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Transformation entails recombination of genes between similar or different lineages, representing a form of bacterial sex that increases standing genetic variation, and the recombination that occurs as part of this process is called chromosomal transformation.

If the incoming ssDNA shares no homology with recipient, but has a self-replication potential , it can be reconstituted into its dsDNA form using recombination and replication function. The recombination that occurs as part of this process is called plasmid transformation or viral transfection(Viret et al., 1991, Chen and Dubnau, 2004, Claveryset al., 2000, Claverys et al., 2009, Dubnau, 1999, Smith et al., 1995, Stewart and Carlson, 1986).

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Chemical transformation method utilizes CaCl2 and heat shock at 42°C to promote DNA entry into cell.

CaCl2 causes the DNA to precipitate onto the surface of the cell.

Perhaps the salt is responsible for some kind of change in the cell wall that improves DNA binding.

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It use a short pulse of electric charge to facilitate DNA uptake.

Electroporation induces formation of microscopic pores within a biological membrane.

These pores allow molecules ions and water to pass from one side of the membrane to other.

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When DNA (the bacterial chromosome) is extracted for

transformation experiments, some breakage into

smaller pieces is inevitable. If two donor genes are

located close together on the chromosome, then there

is a greater chance that they will be carried on the

same piece of transforming DNA and hence willcause a double transformation.

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A DNA library is a collection of DNA clones, gathered together as a source of DNA for sequencing, gene discovery or gene function study.

Among the largest type of DNA library is a Genomic library.

Genomic library which contains the entire Genome (exons and introns).

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The first step in preparing a genomic library ispartial digestion of the DNA by restrictionendonuclease, such that any given sequence willappear in fragment of a range sizes- a range thatis compatible with the cloning vector.

Fragments that are too large or too small forcloning are removed by centrifugation.

The cloning vector such as BAC or YAC plasmid,is cleaved with the same restriction endonucleaseand ligated to the genomic DNA fragments.

The ligated DNA mixture is then used totransform bacterial or yeast cell to produce alibrary of cell types.

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Ideally all the DNA in the genome under studywill be represented in the library.

Transformed bacterium or yeast cell grows into acolony or clone, of identical cells bearing thesame recombinant plasmid.

Cells containing particular DNA sequence can beidentified by DNA hybridization methods.Hybridization method can order individual clonesin library by identifying clones with overlappingsequences.

A well characterized library may containthousands of contigs.

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Generally higher capacity vectors were used for cloning such as cosmids, BACs, PACs, and YACs.

Advantages of such vectors is that the average insert size is much large.

The number of recombinants that need to be screened to identify a particular gene of interest is correspondingly low.

Genes are more likely to be contained within a single clone and fewer clones are required to assemble a conting.

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Principles of Gene Manipulation - S. B. Primrose, Richard M. Twyman, R. W. Old e-book.


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