Tag: lsolation of Specific Human DNA

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

Module Below Start Quiz

Recombinant Dna Technology

RECOMBINANT DNA TECHNOLOGY

Q. 1 Which of the following statements describing restriction endonucleases is TRUE?

 A

They always yield blunt ends

 B

They recognize methylated DNA sequence

 C

They recognize triplet repeats

 D

They cleave both strands in duplex DNA

Q. 1

Which of the following statements describing restriction endonucleases is TRUE?

 A

They always yield blunt ends

 B

They recognize methylated DNA sequence

 C

They recognize triplet repeats

 D

They cleave both strands in duplex DNA

Ans. D

Explanation:

Restriction endonucleases are produced by prokaryotes for cleaving both strands of foreign DNA.
The host cell’s DNA is not degraded because the recognition sites are specifically methylated.
The endonucleases recognize specific short symmetrical sequences known as palindromes.
These cleavage sites contain two fold rotational symmetry in that the sequence is identical but antiparallel in the complementary strands.

In some cases, single-stranded cohesive ends on each of the complementary strands are produced, while in other cases double-stranded blunt ends are formed.
Modern analysis of DNA structure is highly dependent upon the use of different restriction endonucleases that permit the specific hydrolysis of DNA into large polynucleotides. 

Ref: Weil P. (2011). Chapter 39. Molecular Genetics, Recombinant DNA, & Genomic Technology. In D.A. Bender, K.M. Botham, P.A. Weil, P.J. Kennelly, R.K. Murray, V.W. Rodwell (Eds), Harper’s Illustrated Biochemistry, 29e.

Q. 2 After digestion by restriction endonucleases DNA strands can be joined again by which of the following enzymes?

 A

DNA polymerase

 B

DNA ligase

 C

DNA topoisomerase

 D

DNA gyrase

Q. 2

After digestion by restriction endonucleases DNA strands can be joined again by which of the following enzymes?

 A

DNA polymerase

 B

DNA ligase

 C

DNA topoisomerase

 D

DNA gyrase

Ans. B

Explanation:

DNA is cleaved into fragments by restriction endonucleases. After the fragments of DNA have base paired, the ends are covalently joined by the action of DNA ligase

Restriction enzymes in conjunction with DNA ligase can produce vector containing recombinant or hybrid or chimeric DNA.
 
Ref: Essentials of Biochemistry By Naik, 2012, Page 335.

Q. 3 DNA fragments formed by the action of Restriction Endonucleases, are separated by:

 A

Gel electrophoresis

 B

Agarose gel eletrophoresis

 C

Paper Chromatography

 D

High pressure liquid chromatography

Q. 3

DNA fragments formed by the action of Restriction Endonucleases, are separated by:

 A

Gel electrophoresis

 B

Agarose gel eletrophoresis

 C

Paper Chromatography

 D

High pressure liquid chromatography

Ans. B

Explanation:

B i.e. Agarose gel electrophoresis

Restriction endonuclease (restriction enzyme) makes 2 incisions through sugar phosphate backbone (phospho diester bond) of each strand of ds DNAQ at specific sequence k/a recognition sequences or restriction site. So RE restricts viral (bacteriophage – DNA) replicationQ and protects bacteria (prokaryote) from infection by virusQ.

RE cuts both strands of double stranded (ds) DNA at specific restriction sites with palindromic (inverse repeat) arrangement; thus producing smaller, manageable fragments with short sequenes and sticky/ blunt endsQ. These restriction fragments can be isolated by agarose gel /polyacrylamide – electrophoresisQ or HPLC.

After digestion by restriction endonucleases the DNA ends can be ligated (joined/ annealed) by ‘ DNA-ligaseQ

Quiz In Between


Q. 4

In DNA transfer the vectors used from smallest to largest is:

 A

Cosmids, Plasmids, Bacteriophage

 B

Plasmids, Bacteriophage, Cosmids

 C

Bacteriophage, Cosmides, Plasmids

 D

Cosmids, Bacteriophage, Plasmids

Q. 4

In DNA transfer the vectors used from smallest to largest is:

 A

Cosmids, Plasmids, Bacteriophage

 B

Plasmids, Bacteriophage, Cosmids

 C

Bacteriophage, Cosmides, Plasmids

 D

Cosmids, Bacteriophage, Plasmids

Ans. B

Explanation:

B i.e. Plasmid, bacteriophage, cosmid 

*       Plasmid is smallest & most commonly used vectors

*       1Kb = 1000 nucleotide long base

Vector/ Vehicle- DNA

DNA insert size (i.e. can accept DNA fragment of)

Plasmid (PBR 322)

0.01 – 10 kb (smallest)(2

Bacteriophage

(Lambda charon 4A)

10 -20 kb

Cosmids

35 – 50 kb (largest)2


Q. 5 Palindrome is associated with:        

 A

Synthesis of DNA

 B

Extrachromosomal molecule of DNA

 C

Sequence of DNA

 D

Small nuclear RNA

Q. 5

Palindrome is associated with:        

 A

Synthesis of DNA

 B

Extrachromosomal molecule of DNA

 C

Sequence of DNA

 D

Small nuclear RNA

Ans. C

Explanation:

 

Palindromic DNA – A palindrome is a sentence that reads the same forwards and backwards, e.g. ‘Madam I’m Adam’. The DNAs of several eukaryotes are shown to have palindromic sequences, in which nucleotides of one strand going in one direction are the same as the nucleotides of the other strand going in the other direction.

The exact significance of palindromic DNA is not known, although several functions have been suggested.

Short palindromes may function as recognition sites of DNA for proteins which also have a two-fold rotational symmetry, e.g. lac repressor protein, CRP protein and many bacterial restriction enzymes.

Palindromes may also give structural strength to the transcribed RNA by hydrogen bonding in the hairpin loops. If the palindromic sequences are not perfectly symmetrical, imperfect loops may result.

Quiz In Between


Q. 6

DNA amplification is done in by all, except:   
PGI 06

 A

Polymerase chain reaction

 B

Nucleic Acid Sequence Based Amplification

 C

Ligase chain reaction

 D

DNA sequencing

Q. 6

DNA amplification is done in by all, except:   
PGI 06

 A

Polymerase chain reaction

 B

Nucleic Acid Sequence Based Amplification

 C

Ligase chain reaction

 D

DNA sequencing

Ans. D

Explanation:

Ans. DNA sequencing


Q. 7

Nucleic acid amplification techniques are:

 A

PCR

 B

Real time PCR

 C

DNA Cloning

 D

Next generation DNA sequencing

Q. 7

Nucleic acid amplification techniques are:

 A

PCR

 B

Real time PCR

 C

DNA Cloning

 D

Next generation DNA sequencing

Ans. A:B

Explanation:

Ans: a. PCR …, b. Real time ….[Ref Harper 30th/458; Robbins 9th/180; Lippincott 6th/479;Harrison 19th/ 150e-7; http://link. springer. corn]

  • Real-time PCR automates the laborious process of amplifica­tion by quantitating reaction products for each sample in every
  • Cycle.
  • There are several methods for amplification (copying) of small numbers of molecules of nucleic acid to readily detectable levels.
  • These NAATs include PCR, LCR, strand displacement amplification, and self-sustaining sequence replication.
  • The amplified nucleic acid can be detected after the reaction is complete or (in real-time detection) as amplification proceeds. The sensitivity of NAATs is far greater than that of traditional assay methods such as culture.

Quiz In Between



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