Guide Questions from Buckingham

• What is the purpose of denaturation of double-stranded target DNA after electrophoresis and prior to transfer in Southern blot?

  Double-stranded target DNA is denatured to allow hybridization of the probe. Also, single-stranded DNA binds to nitrocellulose filters.
• Name two ways to permanently bind nucleic acid to nitrocellulose following transfer.

  To permanently bind the nucleic acid, bake the filter at 80°C for 30 minutes. Alterna-tively, nucleic acid will be cross-linked to nitrocellulose upon exposure to ultra-violet light.
• If a probe for Southern blot was dissolved in a hybridization buffer that contains 50% formamide, is the stringency of hybridization higher or lower than if there were no formamide?

  The stringeney is higher, since formamide facilitates denaturation of double-stranded DNA.
• If a high concentration of NaCl were added to a hybridization solution, how would the stringency be affected?

  The stringency would be decreased since high salt concentrations exclude nucleic acids, forcing them close together and promoting hybridization.
• Does an increase in temperature from 65°C to 75°Cduring hybridization raise or lower stringency?

  Increasing temperature raises strin-gency. Heat promotes separation of the hydrogen bonds holding the two single strands of double-stranded DNA together.
• At the end of the Southern blot procedure, what would the autoradiogram show if the stringency was too high?

  If the stringency is too high, faint or no bands will be present on the autoradiogram.
• A northern blot is performed on an RNA transcript with the sequence GUAGGUATGUAUUUGGGCGC-GAACCAAAA. The probe sequence is GUAGGUAT-GUAUUUGGGCGCG. Will this probe hybridize to the target transcript?

  This probe will not bind to the target transcript. The sequences are identical and not complementary. Since RNA does not have a complementary partner, the probe must be complementary to the transcript sequence.
• In an array CGH experiment, three test samples were hybridized to three microarray chips. Each chip was spotted with eight gene probes (Genes A-H). Below are results of this assay expressed as the ratio of test DNA to reference DNA. Are any of the eight genes consistently deleted or amplified in the test samples?If so, which ones?

  Gene B is consistently deleted (test/ reference < 1.0)

  
• In an array CGH experiment, three test samples were hybridized to three microarray chips. Each chip was spotted with eight gene probes (Genes A-H). Below are results of this assay expressed as the ratio of test DNA to reference DNA. Are any of the eight genes consistently deleted or amplified in the test samples?If so, which ones?

  Gene E is consistently amplified (test/reference > 1.0).

  
• What are two differences between CGH arrays and expression arrays?

  (a) CGH arrays are performed on DNA.

  Expression arrays are formed on RNA.

  (b) CGH arrays detect deletions and amplifications of chromosomal regions. Expression arrays detect changes in the RNA levels of specific genes.