Everything you need to know about tools and techniques of molecular biology. Some of the most frequently asked questions are as follows:-
Q.1. Name a technique used to separate DNA and RNA molecules according to their size.
Ans: Gel electrophoresis (Fig. 34.1).
Q.2. Why is DNA while subjected to an electric field migrates through the gel towards the positive pole? Flows are the DNA molecules of different size separated? Explain.
Ans: Because DNA is negatively charged DNA molecules are flexible. The pores in the gel matrix give the DNA molecules according to their size (volume). Large molecules migrate more slowly through the gel because they have large effective volume in comparison to small DNA molecules.
Therefore, the large molecules face more interstices of the gel. Once the gel has been run for a given time the molecules of different sizes can be separated because they travel different distances according to the size of the DNA molecules.
On completion of electrophoresis the DNA molecules can be visualized by staining the gel with fluorescent dyes like ethidium which binds to DNA and intercalates between the stacked bases. Each band shows the presence of a population of DNA molecules according to their size.
Q.3. Name the two alternative types of gel matrices. Also give their characteristics.
Ans: These are:
(i) Polyacrylamide and
(ii) Agarose.
The polyacrylamide has got high resolving capability but can separate DNAs over a narrow size range, only. Thus, electrophoresis through polyacrylamide can resolve DNAs which differ from each other in size by as little as single base pair but only with molecules up to several hundred base pairs. On the other hand agarose has less resolving power than polyacrylamide but can separate from one another DNA molecules of up to ten and even hundreds, of kilo-bases.
Very long DNAs are unable to penetrate the pores even in agarose. However, such type of very long DNAs can be resolved from one another if the electric field is applied in pulses which are oriented orthogonally to each other. This technique is called pulse-field gel electrophoresis.