Question

A nonscientist friend of yours asks how findings in a wormorfly can be relevant to human biology. Explain to your friend the importance of model organisms in molecular biology research.

1. Compare and contast one-dimensional SDS-PAGE and two-dimensional gel electrophoresis of proteins.

2. You have purified a protein. When you subject it to SDS-PAGE, two bands are seen. Provide a possible explanation and describe how you could test your hypothesis.

3. Compare and contrast the steps in production of polyclonal and monoclonal antibodies.

4. A chromatographic column in which oligo-dT is linked to an inert substance is useful in separating eukaryotic mRNA from other RNA molecules. On what principle does this column operate?

5. You plan to use the polymerase chain reaction to amplify part of the DNA sequence shown below, using oligonucleotide primers that are hexamers matching the regions shown in red. (In practice, hexamers are too short for most purposes). State the sequence of the primer oligonucleotides that should be used, including their polarity (5?3?), and give the sequence of the DNA molecule that results from amplification.

5?-TAGGCATGCAATGGTAATTTTTCAGGAACCAGGGCCCTTAAGCCGTAGGCAT-3? 3?-ATCGGTACGTTACCATTAAAAACTCCTTGGTCCCGGGAATTCGGCATCGGTA-5?

6. Complete the incomplete diagrams below to show the key structural difference between an NTP, dNTP, and ddNTP (e.g., CTP, dCTP, ddCTP).

What do “d” and “dd” stand for?

Explain why ddNTPs are called "chain terminators" in DNA sequencing reactions.

Answer 1

Ans. Importance of model organism in molecular biology: To understand the life processes it is not possible to monitor the organisms like humans, mammals, cattles etc. as they have a long life span, slow reproductive cycle. Every molecular experiment needs to be observed upto many generations in order to understand the concepts like mutation, variation, heritability etc. The model organisms like worm fly (Drosophilla)has shas short life span and fast breeding. They can produce many reproduce many times in short span of life. Genome size of the wormfly is also simpler and due to which it is more easy to undertand the processes in these orhansisms. They represent as the standar to study the various genetic processes like genetic recombination, replication etc. with more clarity and accuracy.

Ans1. The horizontal electrophoresis is agarose gel electrophoresi, which is used to separate nucleic acid on the basis of molecular size. The current flows in the power unit allow the negatively charged nucleic acid to flow towards anode. The nucleic acid then travels through the agarose and get entrapped according to size, as larger fragments remain up in the gel and the smaller fragment travel longer towards bottom.

On the contrary the SDS PAGE that is sodium dodecy sulphate polyacrylamide gel electrophoresis is a vertical system used to separate various proteins. Proteins are have different nature, positively charged, negatively charged and neutral. They can be of various sizes as well depending upon the amino acids present in the protein. So it mandatory to separate the protein on the basis of charge as well as molecular size in order to get the pure protein.

SDS PAGE is vertical because of two reasons, first it has two gel components stacking gel and running/resolving gel. They are prepared at different time as first resolving gel is allowed to st and then stacking gel of different concentration was poured over it to get stacked above.It is not possible in horizontal system.

Secondly, the polyacrylamide has poor polymerization property in presence of oxygen, so it is recommended to make a gel that is compactly sandwiched between the two plates that allow little or no oxygen to interrupt the polymerization process and hence a good gel will form.

Ans 2: The pure protein when checked on gel splits into two bands. Then the possible reasons are:

1. There is possibility that it might get cleaved by protease during processing that results into two proteins of different sizes.
2. Secondly it is possible that it might be a isoform of protein having similar chemical structure and biological property. It can be confirmed by doing 2D gel electrophoresis.