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Genetic engineering is the manipulation of genetic materials which can be introduced in the host organisms and thus change the phenotype of the host organism. o o o o o o o o o o RESTRICTION ENZYMES DNA LIGASE S1 NUCLEASE REVERSE TRANSCRIPTASE T4 POLYNUCLEATIDE KINASE TAQ POLYMERASE KLENOW FRAGMENT ALKALINE PHOSPHATASE RNase TERMINAL DEOXYNUCLEOTIDE TRANSFERASE FUNCTION The restriction enzymes called ‘’ Molecular scissor’’ and responsible for cutting of DNA Restriction Enzymes belong to a class of enzymes called Nucleases and are of two types: Exonucleases it cleave the DNA molecules at their ends. Endonucleases it cleave the DNA molecules internally at specific recognition sequences. Type 1 Restriction endonucleases are complex endonucleases and have recognition sequences of about 15 bp; they cleave the DNA about 1000bp away 5’-end of the recognition site, e.g. Ecok, EcoB,etc. Type 2 Restriction endonucleases are remarkably stable and induce cleavage either in within or immediately outside their recognition sequences .they require Mg ion for cleavage. Type 3 Restriction endonucleases are intermediate b/w the Type1&Type2 enzymes ; they cleave DNA in the immediate vicinity of their recognition sites, e.g, Ecop1 , Ecop15, Hinf3 etc. FUNCTION It used for ligation of foreign DNA and vectors during gene cloning. It ligate stick ends of DNAs. FUNCTION It removes single- strand protruded from its ends ;both 3’- and 5’-extension FUNCTION It used to produce cDNA ( complementary DNA) copies of , usually , mRNA that are used for creation of cDNA rnase enzymes o FUNCTION Rnase A- it digest RNA but not DNA . RNase H- it also digest RNA and hybrid of RNA-DNA. FUNCTION It is used for addition of phosphate group to an end having a free 5’- OH . It is used for addition of single stranded sequences to 3’-ends of blunt ended fragments. It is used for removing 5’-phosphate group from DNA ends . This enzyme is dimeric glycoprotein with a molecular weight 14,000. It is made up of two identical or similar subunit each of molecular weight 69,000. FUNCTION It is used in polymerase of DNA (in PCR) activity at pH of 9 and temperature around 75C FUNCTION Klenow fragment of E.coli DNA polymerase to make protruding ends doublestranded by extending the strands. o A Vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning . vector must have an origin of DNA replication (ori) that function in the host cell . Any extra-chromosomal small genome .e.g.,plasmid, phage or virus . o o o o o o It should be able to replicate autonomously . A Vector should be ideally less 10 kb in size . The vector should be easy to isolate and purify. It should be easily introduced into the host . The should have a suitable marker genes. A vector should contain unique target site for restriction enzymes. plasmid vector They have high copy no. ( 1 to more than 50 per cell) Plasmid are circular , double-stranded DNA molecules. Plasmid have origin of replication (ori). Plasmid have also antibiotic resistance genes as markers and unique restriction site Cosmid vector Cosmids are essentially a combination of plasmid & lambda phage . They have origin of replication ,unique restriction site, selectable markers from plasmid. They can be used to cloned DNA inserts of upto 40kb. It contain an origin of replication. It accommodate larger DNA insert. It ensure the recombinant phage to be always lytic It contain more than one restriction sites. Some lambda vector contain promoter,operator and lacZ gene of E.coli lac operon. Insertion of DNA fragment in such vectors inactive and act as selectable marker A typical Fish vector is a plasmid.e.g.,pRSV. o Selector Marker, e.g.,ampicillin resistance (amp) o Ori from E. coli plasmid pBR322. o An enhancer/promoter sequence (SV40) o A multiple cloning site for insertion of the DNA insert o A terminal site including polyadenylation site. Drosophila P elements developed as valuable vectors for invaluable genetic material. P elements are transposons of 2.9kb; they contain 3 gene flanked by short (31bp) inverted repeat sequence at their ends. P element vectors are shuttle vector ; like YAC, DNA insert of upto 40kb into P element vector o o o o It is spherical virus with circular, doublestranded 5,243bp chromosome. It encodes 5 protein ,viz, small-T , LargeT,VP1,VP2and VP3. Large –T is essential for viral replication, while VP1,VP2,VP3 form viral capsid. ORI (About 80 bp) It contains pBR322 ori and a selectable marker for cloning in E.coli. Retroviral 5’LTR and 3’LTR are needed for efficient transcription of proviral DNAand for generating 3’end of length transcript. LTRs are essential for intregration of the proviral DNA into host genome. It also has R, U5, U3, P and Pu encoding sequence (involved in reverse transcription). S sequence(needed for splicing to produce functional mRNA for envelope protein synthesis. Psi sequence (necessary for packaging into virions). These Vectors have been designed to replicate in cells of two different species; therefore , they contain two origins of replication , one specific for each host species, as well as those genes necessary for their replication and not provided by host cells . These vector are created by Recombinant DNA techniques . Some of them can be grown in two different prokaryotic species , while others can propagate in a prokaryotic species ,e.g yeast, animal, plants . These vectors can be grown in one host and then moved into another without extra manipulation. OriS, origin of replication from E.coli plasmid. repE , encodes a Rep protein that specifically binds to oriS to initiate replication. DNA insert of upto 300kb Low copy no. These are linear vectors that behave like an yeast chromosome hence they are called (YACs). An ARS sequence for replication CEN4 sequence for centromeric function Telomeric sequence at the ends for protection from exonuclease action. One or two selectable marker gene SUP4, a selctable marker into which DNA insert is intregrated