Recombinant Dna Technology

RECOMBINANT DNA TECHNOLOGY


RECOMBINANT  DNA  TECHNOLOGY

  • when a gene of one species is transferred to another living organism, it is called recombinant DNA technology(genetic engineering).
  • It involves  isolation  and manipulation  of DNA to  make  chimeric or  hybrid DNA.
  • Chimeric (hybrid) DNA is a recombinant DNA containing genes from two different species, e g. molecule containing both human and bacterial DNA sequences.
  • theres are two distinct techniques for introducing foreign genetic material into plant cell genome.

first through vector which requires:

  1. selection and isolataion of the desirable fragment(s) of DNA which contain gene sequences that need to be cloned (known as insert).
  2. generation of recombinanat DNA (rDNA)  by inserting of dNA fragments into carrier DNA molecules termed as VECTOR.
  3. vector–> bacteria like( Agrobacterium tumefaciens, a virus , a plasmid or any other vector) that replicate within host cell
  4. Introduction of rDNA molecules into host cell, direct introduction involves co-cultivation, microinjection.

Recombinant DNA= Vector+ insert(rDNA)

steps are involved in the construction of recombinant DNA –

  1. Fragmentation of DNA by restriction endonuclease enzyme.
  2. Isolation of specific human DNA sequence.
  3. Insertion of isolated human DNA sequence into vector to form chimeric or hybrid DNA molecule.
  4. joining of two different cut DNA fragments by DNA ligase.

Fragmention  of DNA by restriction  endonuclease:-

  1. Most important  tools  in  genetic  engineering  are  enzymes, nzymes is restriction endonuclease.
  2. These are called so because their presence in a given bacterium restricted the growth of foreign (non-bacterial) DNA, e g. bacteriophages.
  3. These cut  DNA  at specific  DNA  sequence  called  its  restriction  site.
  4. These enzymes recognize certain palindromic sequences in the DNA, ie. the short sequence (4-7 base pairs) which has got two fold rotational symmetry.
  5. a short sequence (4-7 bp) of duplex DNA that is same when two strands are read in opposite direction is called palindrome.
  6. Palindrome serves as the target for most restriction endonucleases, Each restriction enzyme (restriction endonuclease) recognizes specific nucleotide sequence, ie. each restriction enzyme has specific palindromic sequence to recognize
  7. methylation of restriction site prevents host DNA digestionby its own restriction nuclease.
  8. If host cell acquires foreign restriction endonuclease (e.g. byhorizontal transfer i.e. conjugation), host DNA is digested by foreign restriction endonuclease because its restriction site is different, which is not methylated in host DNA.
  9. Some restriction enzymes cut both strand at same level, so as to leave no unpaired bases on either end. These ends are called blunt ends.
  10. restriction enzyme cut two strands in staggered manner, leaving two to four nucleotides of one strand unpaired at each end as (cohesive  ends  or  sticky ends or staggered  ends).

lsolation  of Specific  Human DNA:

  • particular fragment of interest can be separated and isolated by gel electrophoresis (most commonly)

lnsertion  of isolated  human  DNA into  vector

  1. Largest  DNA can  be  incorporated  in  cosmid and  shortest  in  plasmid
  2. larger pieces of DNA can be incorporated into bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) and Pt vector.
  3. Plasmid–> pBR 322 < bacteriophase;Lambda charon 4A ( 10-20 kbp) < cosmids (35-50 frp) < BAC = P (50-250 kbp) < YAC (500-3000).
Joining of two  different  cut DNA  fragments:
 
  1. fragments of DNA (one from human genone and another from vector DNA) have base paired, the ends are covalently joined by DNA ligase.

DNA amplification

amplifications of the DNA is required to prepare multiple copies of the DNA, It can be done by: 

  1. Cloning
  2. Polymerase chain reaction (PcR;rrct-our, including real time PCR
  3. Ligase chain reaction (LCR), including Gap LCR
  4. Nucleic acid sequence based amplification (NASBA)

Method  (ii),  (iii)  and  (iv)  are  also  called, nucleic  acid amplification  tests  (NAAT)

Exam Important

  1. It involves  isolation  and manipulation  of DNA to  make  chimeric or  hybrid DNA.\
  2. generation of recombinanat DNA (rDNA)  by inserting of dNA fragments into carrier DNA molecules termed as VECTOR.
  3. vector–> bacteria like( Agrobacterium tumefaciens, a virus , a plasmid or any other vector) that replicate within host cell.
  4. joining of two different cut DNA fragments by DNA ligase.
  5. Palindrome serves as the target for most restriction endonucleases, Each restriction enzyme (restriction endonuclease) recognizes specific nucleotide sequence, ie. each restriction enzyme has specific palindromic sequence to recognize.

amplifications of the DNA is required to prepare multiple copies of the DNA, It can be done by: 

  1. Cloning
  2. Polymerase chain reaction (PcR;rrct-our, including real time PCR
  3. Ligase chain reaction (LCR), including Gap LCR
  4. Nucleic acid sequence based amplification (NASBA)
Don’t Forget to Solve all the previous Year Question asked on RECOMBINANT DNA TECHNOLOGY

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