Please answer the following multiple-part question regarding probe synthesis and
ID: 142526 • Letter: P
Question
Please answer the following multiple-part question regarding probe synthesis and nucleic acid hybridization:
a) You would like to determine how many genes are expressed in the normal hepatocytes (liver parenchymal cells) of a healthy person and the hepatocytes of an alcoholic. Which of the following blots would you utilize and how many hybridization experiments you must perform to complete the experiment?
A. A Sothern blot (membrane-based)/two
B. A Northern blot (membrane-based)/one
C. A microarray/two
D. A gene chip/one
b) Of the two genes, one has a sequence of 5’CCCAUUUGG3’ and another has the sequence 5’CCCAAUUGG3’. You want to distinguish the two genes by Southern blot hybridization. Which of the following parameters you may not have to control?
A. The membrane to be used (nylon membrane versus PVDF membrane)
B. Detergent (i.e. SDS) concentration in the hybridization buffer
C. Presence of non-specific DNA in the hybridization buffer
D. Temperature of the hybridization buffer
c) Solid-phase hybridization is different from solution hybridization because of all these, except______.
A. the former uses probes, the later do not
B. the later may use enzymes, the former don’t
C. the former uses membrane, the later don’t
D. the former immobilize nucleic acids, the later don’t
Explanation / Answer
Ans.
a) D - A gene chip/one
gene chip is a collection of microscopic DNA spots attached to a solid surface. This technique can be used to measure the expression levels of large numbers of genes simultaneously in the normal hepatocytes of a healthy person and the hepatocytes of an alcoholic person. so it would require only one hybridisation experiment.
b)
A. The membrane to be used (nylon membrane versus PVDF membrane)
to distinguish the two genes by Southern blot hybridization, the binding conditions should be tightly controled and non-specific binding should not occur as the two sequences differ at only one base position.
Detergent (i.e. SDS) concentration in the hybridization buffer, Presence of non-specific DNA in the hybridization buffer and the temperature of the hybridization buffer need to be controlled in order to distinguish the two sequences. whereas, the choice of membrane to be used (nylon membrane versus PVDF membrane) will not hinder in distinguishing the two sequences if other three parameters are tightly controlled.
c)
D. the former immobilize nucleic acids, the later don’t