Genomics, DNA Fingerprinting and Gene Editing: CRISPR Notes

The correct answer is B, Alec Jeffreys. Working at the University of Leicester in 1984, Alec Jeffreys found that certain short sequences, the minisatellites or variable number tandem repeats, repeat a different number of times in different people, and that a pattern made from them identifies an individual. His method cut the DNA with restriction enzymes, separated the pieces by gel electrophoresis and revealed them by Southern blotting.

Option A, Kary Mullis, invented the polymerase chain reaction in 1983, the amplification step that later made fingerprinting possible from a few cells, and he shared the 1993 Nobel Prize in Chemistry. Option C, Frederick Sanger, gave the chain-termination method of DNA sequencing, which reads the order of bases rather than comparing repeat patterns. Option D, Hamilton Smith, shared the 1978 Nobel Prize for restriction enzymes. In India the technique was established by Lalji Singh, who is called the father of DNA fingerprinting in India.

The correct answer is B, Emmanuelle Charpentier and Jennifer Doudna. The two scientists showed that the bacterial CRISPR-Cas9 system could be directed by a designed guide RNA to cut any chosen sequence of DNA, which turned a microbial defence mechanism into a general tool for editing genomes, and they shared the Nobel Prize in Chemistry in 2020.

Option A, Watson and Crick, proposed the double helix model in 1953 and shared the 1962 Nobel Prize in Physiology or Medicine with Maurice Wilkins. Option C, Kary Mullis and Michael Smith, shared the 1993 Nobel Prize in Chemistry, Mullis for the polymerase chain reaction and Smith for site-directed mutagenesis. Option D, Werner Arber and Daniel Nathans, shared the 1978 Nobel Prize in Medicine with Hamilton Smith for restriction enzymes. Grouping the prizes by year, 1962, 1978, 1993 and 2020, is the quickest way to hold this chain of discoveries in memory.

The correct answer is B, an endonuclease that cuts both strands of DNA. Cas9 is an enzyme that breaks the sugar-phosphate backbone of both DNA strands at the point where its guide RNA has bound, provided a short adjacent signal called the PAM is present. The cell then repairs the double-strand break, and that repair either knocks the gene out or, when a template is given, writes in the corrected sequence.

Option A describes DNA ligase, which seals fragments together and is used in making recombinant DNA. Option C describes a DNA polymerase such as Taq polymerase, which synthesises a new strand in the polymerase chain reaction; Cas9 copies nothing. Option D describes a vector: plasmids and the Ti plasmid of Agrobacterium tumefaciens carry genes into cells, whereas Cas9 is cargo, not carrier. The two parts of the system are worth separating in memory: the guide RNA chooses the address and Cas9 does the cutting.

The correct answer is B, towards the anode, because DNA is negatively charged. Each nucleotide of DNA carries a phosphate group, and the phosphate backbone gives the whole molecule a net negative charge at the pH used in the laboratory. In an electric field the fragments therefore migrate to the positive electrode, the anode, and the agarose gel slows the longer fragments more than the shorter ones, so the pieces sort themselves by size.

Option A and Option C both place DNA at the cathode, the negative electrode, which would require a positive or neutral molecule; proteins can behave that way at some pH values, but DNA does not. Option D is wrong because separation is exactly what the gel achieves; the dye, such as ethidium bromide, only makes the separated bands visible under ultraviolet light. The small-moves-faster rule is often asked: shorter fragments travel farther from the well.

The correct answer is C, 2003. The Human Genome Project was launched in 1990 as an international public effort coordinated in the United States by the National Institutes of Health and the Department of Energy, with laboratories in several countries taking part. A working draft was announced in 2000 and the project was declared complete in April 2003, the fiftieth year after the double helix was described. It established that the human genome has about three billion base pairs and roughly twenty thousand protein-coding genes.

Option A, 1990, is the year the project began, and it is the commonest wrong choice. Option B, 1998, is the year a private company began a competing sequencing effort, which hurried the public project along. Option D, 2010, belongs to no milestone of the project. India's own genome programmes came later: IndiGen from 2019 and the Genome India Project from January 2020.

The correct answer is B, Taq polymerase. The polymerase chain reaction, invented by Kary Mullis in 1983, repeats three steps: the DNA is denatured near ninety-five degrees Celsius, short primers anneal at a lower temperature, and the strand is extended at about seventy-two degrees. An ordinary polymerase would be destroyed by the heating step, so the reaction uses Taq polymerase from Thermus aquaticus, a bacterium of hot springs, which stays active at these temperatures.

Option A, DNA ligase, joins fragments and is used in recombinant DNA work, not in amplification. Option C, EcoRI, is a restriction enzyme that cuts DNA at a specific sequence; cutting is the opposite of what the reaction needs. Option D, reverse transcriptase, makes DNA from RNA and is used first when the starting material is RNA, as in the reverse transcription test for RNA viruses, but the amplification that follows still needs a heat-stable polymerase.

The correct answer is B, two. In the Watson and Crick model the two strands are held together by hydrogen bonds between complementary bases: adenine pairs with thymine through two hydrogen bonds and guanine pairs with cytosine through three. Because the guanine and cytosine pair has one bond more, a stretch of DNA rich in those two bases needs a higher temperature to separate, a fact used in setting up the polymerase chain reaction.

Option A, one, is too few for any base pair in DNA. Option C, three, is the number for the guanine and cytosine pair, and offering it for adenine and thymine is the standard trap in this question. Option D, four, matches no pair at all. Remember the companion facts as well: adenine and guanine are purines, thymine and cytosine are pyrimidines, and in RNA uracil takes the place of thymine opposite adenine.

The correct answer is B, Hyderabad. The Centre for DNA Fingerprinting and Diagnostics was established in 1995 as an autonomous institution of the Department of Biotechnology and works at Hyderabad on DNA fingerprinting services, diagnostics of genetic disorders and research in genomics. Hyderabad is also where Lalji Singh developed the Bkm-derived probe at the Centre for Cellular and Molecular Biology, which is why the city is associated with the beginnings of the technique in India.

Option A, Pune, hosts the National Centre for Cell Science, a different Department of Biotechnology institution. Option C, Kalyani in West Bengal, is the seat of the National Institute of Biomedical Genomics, which does work on genomics but is not the fingerprinting centre. Option D, Lucknow, has the Central Drug Research Institute and the Indian Institute of Toxicology Research, both under the Council of Scientific and Industrial Research. Pair each institution with its city, as papers ask these matches directly.

The correct answer is B, the Department of Biotechnology. The Genome India Project was launched in January 2020 by the Department of Biotechnology, which works under the Ministry of Science and Technology, and brings together about twenty academic institutions to sequence Indian genomes and build a reference set that reflects the country's genetic diversity. Such a database matters because disease-linked variants found in European populations do not always apply to Indian patients.

Option A, the Indian Council of Medical Research, funds and runs medical research and surveillance but is not the coordinator of this project. Option C, the Indian Space Research Organisation, works on space science and has no role here. Option D, the Central Drug Standard Control Organisation, is the drug regulator that approves medicines and vaccines. Note the companion programme as well: IndiGen, from 2019, under the Council of Scientific and Industrial Research, sequenced a thousand Indian genomes.

The correct answer is B, Bt cotton. Bt cotton carries genes from the soil bacterium Bacillus thuringiensis, which make the plant produce a protein toxic to bollworm larvae. It was approved for commercial cultivation in 2002 and remains the only genetically modified crop cleared for commercial cultivation by farmers in India. Approvals are given by the Genetic Engineering Appraisal Committee under the Ministry of Environment, Forest and Climate Change, while the Review Committee on Genetic Manipulation under the Department of Biotechnology oversees research.

Option A, Bt brinjal, received a clearance that was suspended by a moratorium in 2010 and is not grown commercially. Option C, golden rice, is a vitamin A enriched rice developed abroad and not cleared for cultivation in India. Option D, GM soybean, is grown in some other countries; India allows imports of certain derived oils but not the cultivation of the crop. The examiner's point is the single approved crop and the regulator's name.