Restriction Endonucleases/Enzymes/PCR/DNA
Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites.
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Restriction Endonucleases/Enzymes/PCR/DNA
- 2. Also termed as Restriction Endonucleases OR REases OR RE Functions as Molecular Scissiors/DNA cutters RESTRICTION/DIGESTION The process of restricting OR breaking down the DNA into pieces RESTRICTION DIGEST The product (Fragments of Broken DNA) obtained from restriction Methyl-Transferase/Methylase OR Mtase An enzyme which attaches -CH3 group to host DNA to protect it from REase digestion TERMS TO KNOW
- 3. Also termed as Restriction Endonucleases OR REases OR RE Functions as Molecular Scissiors/DNA cutters RESTRICTION/DIGESTION The process of restricting OR breaking down the DNA into pieces RESTRICTION DIGEST The product (Fragments of Broken DNA) obtained from restriction Methyl-Transferase/Methylase OR Mtase An enzyme which attaches -CH3 group to host DNA to protect it from REase digestion TERMS TO KNOW
- 6. REases can produce Sticky and Blunt ends REases can be Iso-schizo-mers Only Type II restriction endonucleases are the are mostly used Methylated DNA cannot be digested by Reases Some REases also have dual nature (i.g., Reases and Mtases) CHARACTERISTICS OF REase
- 7. REases have specific recognition sequence at which it cuts a DNA molecule For example, the RE called PvuI (isolated from Proteus vulgaris) cuts DNA only at the hexanucleotide 5卒-CGATCG-3卒 PvuII, cuts at a different hexanucleotide in this case 5卒-CAGCTG-3卒 Many RE are Isoschizomers (Different RE but same target sites) REases cleaves Phosphodiestr bonds of Nucleotides SPECIFICITY OF REases Frequency of Recognition Sequence It is calculated mathematically i.e., a tetranucleotide appears every 44=256nt and Hexanucleotide repeated every 46=4096nt Size of restricted DNA fragment Depends upon frequency nt sequence appearance
- 8. Four(four cutters), Five(five cutters), or even longer nucleotide sequences cutting REases are also available e.g., Sau3A (from Staphylococcus aureus strain 3A) recognizes GATC and AlwNI cuts CAGNNNCTG DEGENERATE RECOGNITION SEQUENCES Some REases cut DNA at any one of a family of related sites, e.g., HinfI recognizes GANTC, so cuts at GAATC, GATTC, GAGTC, and GACTC.
- 9. MECHANISM OF RESTRICTON Blunt Ends or Flush End cut Make a simple double-stranded cut in the middle of the recognition sequence HindII, PvuII and AluI are examples of blunt end cutters
- 10. Reases Cut at exactly the same position Usually by two or four nucleotides As bp can stick together again So these are called sticky ends Sticky Ends or Cohesive Ends
- 11. REases with different recognition sequences may produce the same sticky ends BamHI (recognition sequence GGATCC) and BglII (AGATCT) are examplesboth produce GATC sticky ends
- 13. Measure 2 袖g of DNA in 16 袖g of the sample Add required REase (BglII) pure in the mixture Maintain pH 7.4 with Tris-HCl (500mM) Mg+2 (100mM) and Nacl (500mM) for proper activity of RE Add dithiothreitol (DTT) 10mM to stablize and preventing inactivation of RE PERFORMING DIGESTION IN LAB
- 14. Maintain 37属C for 1h Then deactivate REase by exposing it for short time to 70属C or by adding EDTA which binds to Mg+2 of REase and deactivates its function