This set of Bioinformatics Multiple Choice Questions & Answers (MCQs) focuses on “DNA & Genomic Sequencing”.
1. Which of the following is untrue about DNA sequencing methods?
a) Purified fragments of DNA cut from plasmid/phage clones or amplified by polymerase chain reaction (PCR)
b) Clones of DNA fragments are denatured to single strands, and one of the strands is hybridized to an oligonucleotide primer
c) Taq polymerase is quite heat sensitive
d) New strands of DNA are synthesized from the end of the primer
Explanation: In an automated procedure, new strands of DNA are synthesized from the end of the primer by heat-resistant Taq polymerase from a pool of deoxyribonucleotide triphosphates (dNTPs) that includes a small amount of one of four chain-terminating nucleotides (ddNTPs).
2. Using ddATP, the resulting synthesis creates a set of nested DNA fragments, each one ending at one of the as in the sequence through the substitution of a fluorescent-labeled ddATP.
Explanation: A similar set of fragments is made for each of the other three bases. But each set is labeled with a different fluorescent ddNTP.
3. The combined mixture of all labeled DNA fragments is electrophoresed to _____ the fragments by______ and the ladder of fragments is scanned for the presence of each of the four labels.
a) separate, size
b) separate, pH
c) assimilate, pH
d) assimilate, size
Explanation: A computer program then determines the probable order of the bands and predicts the sequence. Depending on the actual procedure being used, one run may generate a reliable sequence of as many as 500 nucleotides.
4. The sequence can also be verified by making an oligonucleotide primer complementary to the distal part of the readable sequence and using it to obtain the sequence of the complementary strand on the original DNA template.
Explanation: For accurate work, a printout of the scan is usually examined for abnormalities that decrease the quality of the sequence, and the sequence may then be edited manually. The first sequence can also be extended by making a second oligonucleotide matching the distal end of the readable sequence and using this primer to read more of the original template.
5. When the process is fully automated, a number of priming sites may be used to obtain sequencing results that give optimal separation of bands in each region of the sequence.
Explanation: By repeating this procedure, both strands of a DNA fragment several kilobases in length can be sequenced. Sequential sequencing of a DNA molecule using oligonucleotide primers is done later.
6. To sequence larger molecules, individual chromosomes are purified and broken into _____ or larger random fragments, which are cloned into vectors designed for large molecules.
Explanation: To sequence larger molecules, such as human chromosomes, individual chromosomes are purified and broken into 100-kb or larger random fragments, which are cloned into vectors designed for large molecules, such as artificial yeast (YAC) or bacterial (BAC) chromosomes. In a laborious procedure, the resulting library is screened for fragments called contigs, which have overlapping or common sequences, to produce an integrated map of the chromosome.
7. Many levels of clone redundancy may be required to build a consensus map because individual clones can have _______
c) two separate fragments
Explanation: Option d here becomes irrelevant as it has quite less relevancy to redundancy of the clones. These do not reflect the correct map and have to be eliminated.
8. Once the correct map has been obtained, unique overlapping clones are chosen for sequencing.
Explanation: However, these molecules are too large for direct sequencing. One procedure for sequencing these clones is to subclone them further into smaller fragments that are of sizes suitable for sequencing, make a map of these clones and then sequence overlapping clones. However, this method is expensive because it requires a great deal of time to keep track of all the subclones.
9. An alternative method is to sequence all the subclones, produce a computer database of the sequences, and then have the computer assemble the sequences from the overlaps that are found.
Explanation: Up to 10 levels of redundancy are used to get around the problem of a small fraction of abnormal clones. This procedure was first used to obtain the sequence of the 4- Mb chromosome of the bacterium Haemophilus influenzae by The Institute of Genetics Research (TIGR) team. Only a few regions could not be joined because of a problem subcloning those regions into plasmids, requiring manual sequencing of these regions from another library of phage subclones.
10. Which of the following is untrue about Shotgun Sequencing?
a) When DNA fragments derived from different chromosomal regions have repeats of the same sequence, they will appear to overlap
b) When DNA fragments derived from different chromosomal regions have repeats of the same sequence, they will appear to scrutinize
c) In a new whole shotgun approach, Celera Genomics is sequencing both ends of DNA fragments of short (2 kb), medium (10 kb), and long (BAC or >100 kb) lengths
d) A large number of reads are then assembled by computer
Explanation: A controversy has arisen as to whether or not the above shotgun sequencing strategy can be applied to genomes with repetitive sequences such as those likely to be encountered in sequencing the human genome. This method has been used to assemble the genome of the fruit fly Drosophila melanogaster after removal of the most highly repetitive regions and also to assemble a significant proportion of the human genome.
Sanfoundry Global Education & Learning Series – Bioinformatics.
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