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Tools in Recombinant Dna Technology

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36 views18 pages

Tools in Recombinant Dna Technology

Uploaded by

manojtbgri5793
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Tools used in recombinant DNA technology

Sreyas s
Tools used in recombinant DNA technology are of 2 types

1 : Enzymes

2 : Vectors
ENZYMES

IT IS OF 3 TYPES

1 : EXONUCLEASE

2 : ENDONUCLEASE

3 : LIGASES
Exonuclease

Exonuclease are the enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain.

That breaks phosphodiester bond either from the 3’ or 5’ end.

It is extensively used in the deletional analysis of promoters to find out which part of the DNA segment can act as a promoter

eg : lamda 5’ exonuclease
Lamda 5’ exonuclease

It has the ability to remove nucleotides one after the other from the 5’ end of the dsDNA either from the blunt end or 5’ over hangs.

It is also used in generating ssDNA and also for the deletional analysis of the promoter elements
ENDONUCLEASE

It is the enzyme that cleave the phosphodiester bond within a polynucleotide chain

RESTRICTION ENDONUCLEASE

In cells restriction endonuclease are used to cut up the invading DNA from viruses.
Restriction sites are ones found in viral DNA.
When a virus injects DNA in to bacterium , the restriction enzymes cut up the viral DNA then it cannot take over the bacterium
Restriction enzymes are classified in to 4 types on the basis of subunit composition , cleavage position ,
sequence specificity and co factor requirements.

1 : type I enzymes

2 : type II enzymes

3 : type III enzymes

4 : type IV enzymes
TYPE I ENZYMES

This enzymes are complex multi subunit , combination of restriction and modification enzymes that cut DNA at randomly far from their recognition sequence.

Type 1 enzymes are of great biochemical interest , but they have little practical value since they do not produce discrete restriction fragment or distinct gel banding patterns
TYPE II ENZYMES

It cut DNA at defined positions close to or within their recognition sequence.

They produce discrete restriction fragment and distinct gel banding patterns , and they are the only class used in the laboratory for routine DNA analysis and gene cloning
TYPE III ENZYMES

Type III enzymes are also large combination restriction and modification enzymes.

They cleave outside of their recognition sequence and require two such sequence in opposite orientation within the same DNA molecule to accomplish cleavage
TYPE IV ENZYMES

Type IV enzymes recognize modified , typically methylated DNA and are


exemplified by the Mcr BC and Mrr systems of E coli
DNA LIGASES

DNA ligases repair single stranded breaks in the double stranded DNA molecule.

It also seals the discontinuities in phosphodiester bonds that arise during replication

enzyme activity occur in the presence of co factor ATP and co enzyme NAD+
RNA DEPENDENT DNA POLYMERASE

It is also called reverse transcriptase


These enzymes have RNA dependent DNA polymerase activity but does not show any 3’-5’ exonuclease activity

It can also perform DNA dependent DNA polymerase activity


Another great feature of this enzyme is that they have RNAse-H activity where the enzymes removes by nickling or displacement of RNA when it is hydrolised to DNA
It can remove nucleotides from both the ends

The alpha subunit has contain both polymerase and RNAse-H activity.this is extensively used in second strand synthesis of cDNA
TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE

It adds dNTPs to the 3’ OH ends of the ssDNA or dsDNA

it is template independent

this can be used for 3’ end labelling

it can be also used for generating 3’ end tails of required nucleotides for cloning purposes
ALKALINE PHOSPHATE

It is available from bacteria and calf intestine

they are used to remove phosphate group from 5’ ends of the ssDNA or from the dsDNA

removal of phosphate group in vectors prevent self ligation

alkaline phosphate from calf intestine can be easily inactivated by heat


POLYMERASE

DNA POLYMERASE I

It is also called kenberg enzyme

it fills the gap in between small DNA segments formed during replication and repair process so it is also called gap filling enzymes

it catalyse polymerisation in 5’-3’ direction , 3’-5’ exonuclease activity , and a unique 5’-3’ exonuclease activity
DNA POLYMERASE II

It is an alternative repair polymerase

it can replicate if the template is damaged

it catalyse exonuclease activity in 3’-5’ direction and polymerisation in 5’-3’ direction


DNA POLYMERASE III

It is participating in normal DNA replication

it catalyse exonuclease activity in 3’-5’ direction and polymerisation activity in 5’-3’ direction

compared to other DNA polymerase it has high polymerase activity.

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