Unit 20: Biotechnology — Short Questions

Restriction endonucleases are molecular scissors — they recognize specific palindromic nucleotide sequences (restriction sites) and cut both strands of DNA there, producing fragments with sticky (complementary single-stranded) ends. They are used to cut out the gene of interest from the source DNA and to open up the vector (e.g., plasmid) at the same site, so that compatible sticky ends are produced on both. DNA ligase is the molecular glue — it joins the gene of interest to the cleaved vector by forming phosphodiester linkages between adjacent nucleotides, sealing the two pieces into a single recombinant DNA (vector) molecule.

Vectors (molecular carriers such as plasmids, bacteriophages, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes, P1 artificial chromosomes, and animal viruses) carry the gene of interest into host cells. The gene of interest is attached to the vector, converting it into a recombinant DNA molecule (rDNA), which is then introduced into a suitable host (expression system) where it is cloned and/or expressed.

The gene of interest can be obtained in three ways: (i) direct synthesis of the gene in the lab using enzymes such as polymerases and ligases; (ii) synthesis by reverse transcription of its mRNA using reverse transcriptase to produce complementary DNA; (iii) cleavage from the chromosome using restriction endonucleases, which recognize a specific restriction site and cut both DNA strands there, producing the gene fragment with sticky ends so it can be isolated.

Step 1, DNA cleavage the source DNA (containing the gene of interest) and the vector DNA (e.g., plasmid) are each cleaved using the same restriction endonuclease, producing compatible sticky ends on the gene of interest and on the cleaved vector. Step 2, production of recombinant DNA: the cleaved vector and the gene of interest are incubated together in the presence of DNA ligase, which forms phosphodiester linkages joining them, converting the vector into a recombinant vector carrying the gene of interest.

A restriction endonuclease is a molecular scissor that recognizes a specific nucleotide sequence (restriction site) and cuts both strands of DNA there, producing fragments with sticky ends. DNA ligase, in contrast, is a molecular glue that joins two DNA fragments together by forming phosphodiester linkages between adjacent nucleotides. Thus, an endonuclease cuts DNA while ligase joins DNA.

PCR is an in vitro technique that rapidly makes multiple identical copies of a specific DNA segment using primers, nucleotides and Taq polymerase through repeated cycles of denaturation, annealing and extension; it does not require a living host cell. Recombinant DNA technology, on the other hand, involves cutting the gene of interest and a vector with restriction endonucleases, joining them with DNA ligase to form recombinant DNA, and introducing this into a living host (e.g., bacteria) where it is cloned and/or expressed as the host multiplies.

PCR is used (i) as an efficient diagnostic technique to detect genotypes of infectious agents; (ii) to detect genetic mutations responsible for certain genetic diseases and cancers; (iii) in criminal investigations to prepare DNA fingerprints from tiny samples such as dried blood or a single hair; (iv) by physicians to detect genetic defects in very early embryos by amplifying DNA from a few sloughed-off cells; and (v) to examine the DNA of extinct species, as long as even a minuscule amount of intact DNA remains. It is also used to build genomic libraries for DNA sequencing.