7.1.4 Biotechnology: Detection of Cell Clones

Question 1

note

Answer 1

• As bacteria containing plasmids with inserted genes multiply, cell clones are produced. Clones must be screened in order to determine which clones have plasmids containing the gene of interest.
  • A shotgun approach to detecting bacteria for a gene of interest involves testing each colony for the presence of the gene or gene product of interest
• Cells that are being screened for the gene of interest must be cultured and then transferred to a filter. The cells must then be denatured to expose the DNA and separate the complementary strands of the DNA.
• The DNA on the filter is then exposed to the DNA probe. If the probe matches the sequence on the filter, then it will hybridize to the DNA by complementary base pairing. The filter must then be rinsed to wash off all excess unhybridized probe.
• The filter is then exposed to a photographic film, which shows spots where it has been exposed to radioactive probes. The images on the film correspond to cells that contain the gene of interest.

Question 2

DNA probe

Answer 2

• A DNA probe is a synthesized segment of radioactively labeled DNA (or RNA) used to find a gene of interest. Radioactive DNA probes can be constructed for the detection of specific DNA sequences within cells. This method can be used to screen bacteria for the presence of a particular gene.
  • A DNA probe will hybridize with a complementary sequence of DNA thus allowing it to be distinguished from other DNA sequences.
• A DNA probe for the gene of interest can be constructed using DNA segments, which contain radioisotopes. Radioisotopes give off radiation that can be detected on film.
• The sequence of a portion of the gene of interest must be known in order to construct a probe segment that will base-pair with a single strand of DNA

Question 3

Place the following steps for gene detection in the correct order (does not include every step).

i. add labeled probe
ii. treat cells to denature DNA
iii. culture bacterial colonies
iv. transfer cells to filter
v. autoradiography

Answer 3

• iii, iv, ii, i, v

Question 4

Gene cloning is useful for

Answer 4

• producing proteins products.

- creating DNA probes that can be used to identify similar genes.

Question 5

Which of the following statements about nucleic acid probes is incorrect?

Answer 5

• They are double-stranded.

Question 6

In terms of DNA technology, a probe is

Answer 6

• a piece of DNA complementary to the gene of interest

Question 7

One effective but inefficient method of determining whether a particular bacterial colony contains a specific gene of interest is

Answer 7

• testing for the protein product of the gene of interest