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Enzymes for DNA Manipulationin Recombinant DNA

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Other Modification Enzymes:End-modification enzymes

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Alkaline Phosphatase

Removes 5' phosphate groups from DNA andRNA

Also remove phosphates from nucleotides andproteins

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Examples: Bacterial alkaline phosphatase BA!": the most active

$ut also the most difficult to destro% at the end of thedephosphor%lation reaction

&alf intestinal alkaline phosphatase &!": purifiedfrom $ovine intestine# less active than BA!# $ut most

(idel% used in molecular $iolog% $ecause it can $e

effectivel% destro%ed $% protease digestion or heat )5for *+ minutes in the presence of 5 m, ED-A"

.hrimp alkaline phosphatase .A!" is derived fromcold/(ater shrimp# destro%ed $% heat 05& for *5minutes"

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1ses for alkaline phosphatase in DNAmanipulations:

Removing 5' phosphates from plasmid and$acteriophage vectors that have $een cut (ith arestriction en2%me3-his prevents self/ligation ofthe vector and there$% greatl% facilitates ligationof other DNA fragments into the vector

Removing 5' phosphates from fragments of DNAprior to la$eling (ith radioactive phosphate

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En2%me that catal%2es the transfer of aphosphate from A-! to the 5' end of either DNAor RNA

Polynucleotide inase !PN"

-he target nucleotide is lacking a 5'phosphate either $ecause it has $eendephorphor%lated or has $een

s%nthesi2ed chemicall%

DNA or RNA that has a 5' phosphatincu$ated (ith an excess of AD!# !first transfer the phosphate from th

nucleic acid onto an AD!# forming Aleaving a dephosphor%lated target

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1ses for pol%nucleotide kinase in DNAmanipulations: !hosphor%lating linkers and adaptors# or

fragments of DNA as a prelude to ligation# (hichreuires a 5' phosphate

Radiola$eling oligonucleotides# usuall% (ith 67!#for use as h%$ridi2ation pro$es

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#erminal deo$ynucleotidyl transferase

8$tained from calf th%mus tissue

A template/independent DNA pol%merase: a$leto s%nthesi2e a ne( DNA pol%nucleotide (ithout

$ase/pairing of the incoming nucleotides to anexisting strand of DNA or RNA

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1ses for -erminal deox%nucleotid%l transferasein DNA manipulations: 9omopol%mer tailing: converting $lunt ends

into stick% ends

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End of %ecture hank 'ou