Chapter 41: Molecular Biology Tools
You are carrying out experiments designed to utilize DNA encoding a wild-type gene to treat a disease resulting from inheritance of a defective gene. Which of the following techniques would be the most useful in pursuit of the goal of these studies?
B. polymerase chain reaction, PCR
C. restriction fragment length polymorphism, RFLP
Answer A: Transgenesis with humans would allow for the elimination of disease genes in a population of offspring; however, technical as well as ethical issues likely will prevent any transgenic experiments to be carried out with human eggs. Therefore, the ability to replace known disease genes with normal copies in afflicted humans is the ultimate goal of gene therapy. Human gene therapy protocols aim to introduce correcting copies of disease genes into somatic cells of the affected individual. Expression of a correct copy of an affected gene in somatic cells prevents transmission through the germ line, thereby avoiding many of the ethical issues of transgenesis. This is analogous to treatment of individuals by organ or tissue transplantation.
You wish to analyze the pattern of genes expressed in a breast cancer in order to identify potential differences to normal breast tissue cells. Which of the following enzymes is most useful to you in your anticipated studies?
E. restriction endonucleases
Answer D: One of the most effective ways to ascertain gene expression differences between 2 different cell types is to analyze the mRNA differences. This can be done by a number of different techniques, the most rapid of which would be with the use of gene microarrays. In order to successfully utilize gene microarrays requires that the mRNAs first be converted into cDNAs with the use of reverse transcriptase.
You wish to incorporate radioactive phosphate onto the 5′-ends of a fragment of DNA so that it can be used as a probe on a Southern blot of genomic DNA. Which of the following enzymes is required in order for you to carry out the labeling of the DNA with radioactive phosphate?