ENDONUCLEASES Enzyme which produced single double stranded cut within DNA molecule (that is they do not digest DNA molecules from their end)

Why need the modern biotechnology(manipulate the DNA) Uses of the molecular biotechnology Technical development in the PCRTechnical development in the PCR In the several manufacturing processIn the several manufacturing process Different Enzyme use in the manipulation, different purpose Protease(detergents)Protease(detergents) Glucose oxidase(glucose to gluconic acid, industrial chemical, diagonastic blood sugar)Glucose oxidase(glucose to gluconic acid, industrial chemical, diagonastic blood sugar) Renin(cheese)Renin(cheese) papain(papaya plant, animal feed)papain(papaya plant, animal feed) Glucose isomerase(glucose to fractose)Glucose isomerase(glucose to fractose) papsin(animal protease, in manufacturingpapsin(animal protease, in manufacturingIndustry

urokinase(anti-trombiontic agent)urokinase(anti-trombiontic agent) asperaginase(cancer chemotherapy)asperaginase(cancer chemotherapy) Tyrosine hydroxylase(parkansons disease)Tyrosine hydroxylase(parkansons disease) Enzymes use to manipulate the DNA, recombinant  Restriction endonucleases\knives\scissors\scalpal Type of restriction enzyme Type1 Not useful for gene manipulationNot useful for gene manipulation Interact with unmodified recognition sequenceInteract with unmodified recognition sequence Traveling distance npTraveling distance np Cleavage only one strand of DNA at random siteCleavage only one strand of DNA at random site Create a gap of 75n in lengthCreate a gap of 75n in length Cleavage site is non-specificCleavage site is non-specific

Type11 Useful for gene manipulationUseful for gene manipulation Recognized a particular DNA sequenceRecognized a particular DNA sequence Mg ion for recognition for restrictionMg ion for recognition for restrictionType111 They require for ATP, mg ionThey require for ATP, mg ion They have intermediate properties between type1 and type11They have intermediate properties between type1 and type11 Naming of restriction enzyme Eco r1( eco spp name, genus name general name,1 one enzyme have more then one restriction enzyme) Target sit e of restriction endonuclease Nature of the cut end Blunt end styleBlunt end style (a) gg cc (a) gg cc cc gg cc gg Cut across both strand of DNA Join both strand without introducing material

Sticky end styleSticky end style (b) GAA TTC CTT AAG CTT AAG (b2) G AATTA CTTAA G CTTAA G Cut on the single strand Isoschizomers (restriction enzyme) isolate from different organism but recognized identical base sequence e.g. Asp 718(Achromobactor spp) and Kpn1(Klebiella pneumoniae) g/gtaccc g/gtaccc ccatg\g ccatg\g

Ligases\sutures Involved in the repairing of the DNA Sealing and reunion of the DNA Take part in the DNA replication and recombination Production of the hybrid DNA( genetic engineering) Seal the nick chain Activity of ligases 1.Sticky end ligation 5 pGATCC G pGATCC G 3 5 pGATCC G pGATCC G 3 3 G CCTAGp G------CCTAGp 5 3 G CCTAGp G------CCTAGp 5 DNA ligase + ATP DNA ligase + ATP 5 pGATCC GGATCC G 3 3 G CCTAGG CCTAGp 5

Pairing of fragment is the transient Weak hydrogen boding Fragment is stabilized by formation of covalent bond b\t 5 phosphoryl one strand and 3 hydroxyl on the other strand 2.Blunt ended ligations blunt DNA can be ligatedblunt DNA can be ligated There is no base paring hold fragment together temporarilyThere is no base paring hold fragment together temporarily Blunt end is the useful for joining together DNA fragment which are not produced by same restriction enzymeBlunt end is the useful for joining together DNA fragment which are not produced by same restriction enzyme Cleavage with hind 111 cleavage with Ecori A 3 5 pAATTC A 3 5 pAATTC TTCGAp 5 3 G TTCGAp 5 3 G s1 nuclease (which digest single strand) s1 nuclease (which digest single strand) A 3 5 pC TP5 3 G DNA ligase (high concentration) +ATP DNA ligase (high concentration) +ATP AC AC TG TG

S1 nucleases S1 nucleases Analyze DNA-RNA hybrid structure to map transcript It is use for DNA mapping Hairpin lop structure formed during synthesis of cDNA digested by this enzyme DNA polymerase 1,holoenzyme 1. Bifunctional activity (5 exonucleatic activity in addition to DNA synthesis) 5 exonuclease activity degrade the DNA strand which complementary to the template strand and thus forming a nick 5 exonuclease activity degrade the DNA strand which complementary to the template strand and thus forming a nick DNA synthesis began at 3end of the nick and produce new strand of the DNA complementary to the templateDNA synthesis began at 3end of the nick and produce new strand of the DNA complementary to the template 2. Prepare labeled DNA of high specific activity 3. Catalyze de novo DNA synthesis

DNA polymerase 1,klenow fragment DNA polymerase 1+protease two protein fragmentDNA polymerase 1+protease two protein fragment Large fragment is called klenow fragment Large fragment is called klenow fragment Dose not exhibit 5 exonuclease activityDose not exhibit 5 exonuclease activity Use to synthesized DNA when there is no need of removing DNA strand which is complementary to template strandUse to synthesized DNA when there is no need of removing DNA strand which is complementary to template strand For production of second strand of cDNAsFor production of second strand of cDNAs T4 DNA polymerase Lake 5exonuclease activityLake 5exonuclease activity active in 3 exonuclease activity active in 3 exonuclease activity (It act on both 3 end of double stranded DNA resulting in 5 single stranded extension dna complementry to these single strand are synthesis with deoxynucleoside triphosphase enzyme) (It act on both 3 end of double stranded DNA resulting in 5 single stranded extension dna complementry to these single strand are synthesis with deoxynucleoside triphosphase enzyme) Useful in generating 5 single strand endUseful in generating 5 single strand end

Tag DNA polymerase (thermus aquaticus) It lacks 5 to 3 and 3 to5 exonucleatic activityIt lacks 5 to 3 and 3 to5 exonucleatic activity It is ideal for both automated and manual DNAIt is ideal for both automated and manual DNA sequencing (because fast, highly progressive little sequencing (because fast, highly progressive little or no 3-exonucleatic activity It is use in the PCR and can withstand high temperature and DNA sequencingIt is use in the PCR and can withstand high temperature and DNA sequencing Ribonuclease (RNAase H) Is an endonuclease that degrade RNA portion of the RNA-DNA hybridIs an endonuclease that degrade RNA portion of the RNA-DNA hybrid Key enzyme in the cDNA cloning procedureKey enzyme in the cDNA cloning procedure Use to detect the DNA-RNA hybridUse to detect the DNA-RNA hybrid Use to remove the poly A tail from mRNAUse to remove the poly A tail from mRNA

Reverse transcriptase RNA dependent DNA polymeraseRNA dependent DNA polymerase Enzyme require DNA primer complementary to RNAEnzyme require DNA primer complementary to RNA template template Mg and Mn for initiation of transcriptionMg and Mn for initiation of transcription In vitro synthesis of complementary DNA from mRNAIn vitro synthesis of complementary DNA from mRNA It is also contain DNA dependent DNA polymeraseIt is also contain DNA dependent DNA polymerase Activity responsible for second strand formation in cDNA Activity responsible for second strand formation in cDNA Reverse transcription mediate the conversion of genetic information mRNA molecule to d strand DNA moleculeReverse transcription mediate the conversion of genetic information mRNA molecule to d strand DNA molecule Poly (A) polymerase In vitro gene manipulationIn vitro gene manipulation Catalyze the addition AMP unit to the 3 end of RNACatalyze the addition AMP unit to the 3 end of RNA

Deoxyribonuclease 1  DNase 1 is an end nuclease which digest either single or double stranded DNA.  Addition of mg +2 ensure random cleavage mn+2 gives cleavage nearly at the same place on both strand  enzyme are enable the scientist to design and engineer the gene and thus produce desire specification