Eukaryotic Vehicles | Passenger DNA | Foregn DNA | DNA Insert

Eukaryotic Vehicles

• We have so far described vectors that are suitable for prokaryotic cells especially in E. coli.
• The vehicles (DNAs) are constructed in various ways , but the DNA of Simian virus 40 (SV 40) or its derivative is most commonly used vehicle for mammalian cells.
• This vehicle can accept an insert of a length of 4.3 Kb and it does not contain any marker

Passenger DNA / Foreign DNA / DNA Insert

• The DNA which is transferred from one organism into another by joining it with the vehicle DNA, is called passenger or foreign DNA.
• Generally three types of passenger DNAs are used.
• These are complementary DNA (cDNA), synthetic DNA (sDNA) and random DNA.

Complementary DNA ( cDNA ).

• It is synthesized on RNA template (usually mRNA) with the help of reverse transcriptase enzyme discovered by Temin and Baltimore in 1970 and essential nucleotides.
• The DNA is separated from the RNA - DNA complex in the presence of alkaline phos phatase enzyme.
• A cDNA strand is formed on the separated single- stranded DNA template with the help of DNA polymerase enzyme.

Synthetic DNA (DNA)

• It is synthesized on DNA template or without a template
• Artificial Synthesis of DNA on Template

• Kornberg and his coworkers (1959) produced DNA from deoxyribonucleoside triphosphates in the presence of DNA polymerase enzyme, metal ions and a segment of viral DNA which acts as a primer.
• Artificial Synthesis of DNA without a Template

• Hargobind Khorana and his coworkers in 1970, synthesized the gene which was responsible for coding for tyrosine tRNA of E. coli.
• The gene had 207 nucleotide pairs.

Random DNA

• Small fragments are formed by breaking a chromosome of an organism in the presence of restriction endonucleases,

Measurement of DNA

• DNA in a cell is measured in terms of DNA content in 1C (G, phase of cell cycle) cells genome size in a haploid cell.
• DNA content in a 1C human cells is 3.2 picograms.
• DNA size in a haploid human cell is 3.2 x 109 bp

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