Recombinant Dna Technology

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. 

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. 

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

B i.e. Plasmid, bacteriophage, cosmid 

*       Plasmid is smallest & most commonly used vectors

*       1Kb = 1000 nucleotide long base

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.

Ans. DNA sequencing

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.