7.2.2 Using Reverse Transcriptase to Make cDNA

Question 1

note

Answer 1

• review: During transcription, a strand of RNA is synthesized from a DNA template. In eukaryotes, non-coding regions called introns must be removed from the RNA strand in order for it to code for a functional protein.
• As a lab technique, reverse transcriptase can be used to make DNA from RNA. The DNA that results from an RNA template is called complementary DNA or cDNA. cDNA is much shorter than the original gene because it lacks introns.
• The gene for the clotting protein, factor VIII, is made up of 186,000 base pairs. The RNA that it codes for has 9000 base pairs. Part of the reason that the actual gene has so many more base pairs than the RNA is because of the presence of introns.

Question 2

DNA microarray assay

Answer 2

• researchers can test for gene expression in a particular tissue
• In a DNA microarray assay, researchers test for the genes expressed in a particular tissue. RNA is first extracted from a particular type of tissue and labeled with a fluorescent dye. The labeled RNA segments are added to the DNA microarray (DNA fragments from specific genes fixed to a slide). If the sequences are complementary, the RNA/DNA complex will fluoresce. Researchers have thus traced the RNA to the appropriate gene.

Question 3

cDNA library

Answer 3

• a collection of many bacterial cell clones, each containing a gene that was expressed in the tissue from
  which mRNA was collected
• The RNA from the tissue can also be used to make cDNA. Each cDNA that is produced from RNA can be placed into a plasmid and put into a bacterium to make a cDNA library. The library produced will contain only genes expressed in the particular tissue from which it was constructed.

Question 4

reverse transcriptase

Answer 4

• can be used to make a DNA segment from RNA (as shown on the left). The DNA segment produced is called complementary DNA or cDNA. Complementary DNA is much shorter than the original gene because it lacks introns. It can be cloned more easily in bacterial cells because they lack the ability to remove introns
  from RNA transcripts.

Question 5

If a gene from a eukaryotic cell were cloned directly from its chromosomal DNA, how much gene product would you expect to see produced by bacterial colonies containing the foreign DNA?

Answer 5

• None

Question 6

cDNA is

Answer 6

• single-stranded DNA identical to the coding sequences of the chromosomal DNA.
• single-stranded DNA complementary to the mRNA
• B and C

Question 7

Which of the following are necessary to make cDNA?

i. reverse transcriptase
ii. nucleotides
iii. DNA polymerase
iv. mRNA
v. gene product

Answer 7

• i, ii, iv

Question 8

While the DNA sequence necessary for the production of a particular protein in prokaryotes can be determined directly from the chromosomal DNA, this is not possible in eukaryotes. The reason for this is that

Answer 8

• eukaryotic mRNA is spliced prior to translation

Question 9

Factor VIII is a eukaryotic gene product. Which of the following correctly matches the macromolecule with its length in nucleotides or amino acids?

Answer 9

• DNA 186,000
• mRNA 9,000
• protein 2,351