Modifying enzymes .pptx

The enzymes that effect change in the DNA chemical constitution or topology are generally referred to as DNA modifying enzymes.
All enzymes involved in genetic engineering (the degradation, synthesis and alteration of the nucleic acids) fall under the broad category of enzymes known as DNA modifying enzymes.

1. Modifying enzymes used in molecular cloning Elizabeth Philip

2. DNA MODIFYING ENZYMES The enzymes that effect change in the DNA chemical constitution or topology are generally referred to as DNA modifying enzymes. All enzymes involved in genetic engineering (the degradation, synthesis and alteration of the nucleic acids) fall under the broad category of enzymes known as DNA modifying enzymes.

3. 1. DNA Methylases Addition of methyl groups (methylation) to the nucleotides in a DNA molecule. In prokaryotes, the major role of methylation is to protect host DNA against degradation by restriction enzymes. They generally catalyze the transfer of methyl g roups from S-adenosyl-methionine (SAM) to specific nucleotides of double stranded DNA molecules.

4. Dam Methylases : The methylase encoded by the dam gene (dam methylase) transfers a methyl group from SAM ( S-adenosyl-methionine ) to the N6 position of the adenine base in the sequence 5' … GATC … 3’. Dcm Methylases : The methylase encoded by the dcm gene ( dcm methylase) methylates the internal cytosine base, at the C5 position, in the sequences 5' … CCAGG … 3' and 5' … CCTGG … 3'. Dam and Dcm methylation:

5. 2. Ligases Enzymes that join the nucleic acid molecules together.  Catalyses the formation of a phosphodiester bond between the 5' phosphate of one strand and the 3' hydroxyl group of another.  Function: to repair single strand breaks (discontinuities) that arise as a result of DNA replication and/or recombination. In a sense, they are the opposite of restriction endonucleases, but they do not appear to be influenced by the local sequence. Ligases require either ATP or NAD+ as a cofactor, and this contrasts with restriction endonucleases.

6. a) E. Co li DNA ligase Will catalyze a phosphodiester bond between duplex DNA containing cohesive ends. It will not efficiently ligate blunt ended fragments. Requires NAD+ as a cofactor. b)T4 DNA ligase Isolated from bacteriophage T4. Will ligate the ends of duplex DNA or RNA. This enzyme will join blunt-end termini as well as ends with cohesive (complementary) overhanging ends (either 3' or 5' complementary overhangs). This enzyme will also repair single stranded nicks in duplex DNA, RNA or DNA/RNA duplexes. Requires ATP as a cofactor.

7. 3.Phosphatases Alkaline phosphatase is purified from either E. Coli or higher organisms (e.g. calf intestine). It is used for removal of 5′-phosphate groups from nucleic acids in order to prevent recircularization of DNA vectors in cloning experiments. Now, this vector can be joined with the foreign DNA and the nicks formed can be ligated using ligase. This vector containing the insert is suitable for transformation

8. Cutting dna with restriction enzyme Free phosphate & free hydroxyl groups Alkaline phosphatase

9. Calf intestinal phosphatase (CIP): Also c atalyzes the removal of 5' phosphate groups from RNA, DNA and ribo - and deoxyribo - nucleoside triphosphates (e.g. ATP, rATP ). CIP treated duplex DNA cannot self ligate. Hemi-phosphorylated duplexes will be ligated on one strand (the phosphorylated strand) and remain "nicked" on the other . Bacterial Alkaline phosphatase ( BAP): E. coli alkaline phosphatase catalyzes the removal of 5’-phosphate groups from DNA, RNA, and ribo - and deoxyribonucleoside triphosphates. It can be used to prevent self-ligation of vectors because alkaline phosphate-treated DNA fragments lack the 5’-phosphoryl termini required for the actions of DNA ligases.

10. 4 . Polynucleotide kinase P roduct of the T4 bacteriophage PNK transfers the gamma phosphate from ATP to the 5' end of a polynucleotide (DNA or RNA). The target nucleotide is lacking a 5' phosphate either because it has been dephosphorylated or has been synthesized chemically.  The resulting product could be used to end-label DNA or RNA, or in a ligation reactions..