Chp 4 Manipulation of DNA Huseyin Tombuloglu PhD. GBE310, Spring 2015.

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Chp 4 Manipulation of DNA Huseyin Tombuloglu PhD. GBE310, Spring 2015

Chp 4 Manipulation of DNA

Basic steps of gene cloning

Nucleases are enzymes that cut, shorten, or degrade nucleic acid molecules Exonucleases remove nucleotides one at a time from the end of a DNA molecule. Endonucleases are able to break internal phosphodiester bonds within a DNA molecule.

Ligases join nucleic acid molecules together.

DNA Polymerases

DNA polymerase I is an example of an enzyme with a dual activity DNA polymerization DNA degradation.

Klenow fragment (have no nuclease activity) The polymerase and nuclease activities of DNA polymerase I are controlled by different parts of the enzyme molecule. The nuclease activity is contained in the first 323 amino acids of the polypeptide, so removal of this segment leaves a modified enzyme that retains the polymerase function but is unable to degrade DNA. The major application of these polymerases is in DNA sequencing

Reverse Transcriptase an enzyme involved in the replication of several kinds of virus It is used to complementary DNA (cDNA) synthesis (or cloning)

Modifying Enzymes

The discovery and function of restriction endonucleases 2500 different ones have been isolated and more than 300 are available for use in the laboratory The discovery of these enzymes, which led to Nobel Prizes for W. Arber, H. Smith, and D. Nathans in 1978

Type II Endonucleases (restriction endonucleases) A particular enzyme cleaves DNA at the recognition sequence and nowhere else. PvuI (isolated from Proteus vulgaris) CGATCG PvuII CAGCTG

Blunt ends and sticky ends

Most restriction endonucleases function at pH 7.4, all type II restriction endonucleases require Mg2+ in order to function. dithiothreitol (DTT), stabilizes the enzyme and prevents its inactivation. Providing the right conditions for the enzyme is very important— incorrect NaCl or Mg2+ concentrations not only decrease the activity of the restriction endonuclease, they might also cause changes in the specificity of the enzyme

Sticky ends ligation is efficient than blunt end. Less DNA is enough for DNA pair matching

Putting sticky ends onto a blunt-ended molecule X A common situation is where the vector molecule has sticky ends, but the DNA fragments to be cloned are blunt-ended. three methods can be used to put the correct sticky ends onto the DNA fragments 1- Linkers 2- Adaptors 3- Blunt end ligation with topoisomerases

Possible restriction of this method: Generally your target DNA includes RE clevage site 1- Linkers

2- Adaptors

Producing sticky ends by homopolymer tailing

3- Blunt end ligation with topoisomerases

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