This set of Molecular Biology Multiple Choice Questions & Answers (MCQs) focuses on “Automated Sequencing”.
1. Automated sequencing is defined as _________
a) Chain termination sequencing
b) Radio labeled sequencing
c) Real time fluorescence sequencing
Explanation: To automate the process of sequencing DNA it is desirable to acquire sequence data in real time. This is done by attaching fluorescent tags to the chain terminating nucleotides and then detecting the bands within the gel during the electrophoretic separation.
2. Automated sequencing uses _____________
a) Enzymatic process
c) Chain termination
d) Fluorescent tagging
Explanation: Automated sequencing uses fluorescent tagging method for the detection of bands. These tags are attached to the chain terminating nucleotides. Each of the four dideoxynucleotide carries a different flurophore.
3. No chain termination takes place in automated sequencing.
Explanation: As the tags are attached to the four dideoxynucleotide polymerase cannot synthesize after that. Thus incorporation of the tags ensures the termination of the synthesis.
4. As different fluorescent tags affect the mobility of fragments thus only one type of tag is used.
Explanation: The four dideoxynucleotide carries a different flurophore to simplify the base identification. As different fluorescent tags affect the mobility of fragments thus sophisticated software is incorporated into the scanning process to ensure that the bands are read in the correct order.
5. With respect to automated sequencing which of the following is true?
a) Detected by radioactive tags
b) Multiple plane sequencing
c) Horizontal scanning one sequence per lane
d) Vertical reading software incorporation
Explanation: By using four different fluorescent dyes, it is possible to electrophorese all four chain terminating reactions together in one plane of a sequencing gel. The DNA bands are detected by their fluorescence as they electrophorese past the detector. In the detector, it is made to scan horizontally across the base of a slab gel.
6. Which of the following feature is required for the high sensitivity of the tags used for autosequencing?
a) Strong absorption at UV spectrum
b) Maximum emittion at different wavelengths
c) Different mobility shifts
d) Induce circularization of DNA fragments
Explanation: For high sensitivity DNA detection in four colours sequencing the following criteria must be met:
i) Each of the four dyes must exhibit strong absorption at the common laser wavelength.
ii) To have an emission maximum at a distinctly different wavelength.
iii) To introduce the same relative mobility shift of the sequencing fragments.
7. Automated sequencing is a real time one trial method of sequencing and is highly handy.
Explanation: The use of single gel track for all four dideoxy reactions means that the problem is less acute in automated sequencing than manual sequencing. It is desirable to sequence a piece of DNA several times and on both strands, to eliminate caused by technical problems.
8. A third generation sequencing can sequence upto ____________nucleotides per day.
Explanation: The third generation sequencing can sequence upto 750000 nucleotides per day. These new generation sequencers uses a 96 capillary sequencer filled with the gel matrix.
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