6. Probe Based Nucleic Acid Technique

What is a probe in nucleic acid techniques?

A probe is a single stranded sequence of DNA or RNA used to search for its complementary sequence in a sample genome.

What techniques commonly use probes?

Southern Blot, Fluorescence in situ hybridization (FISH), Microarray.

What does a Southern blot detect?

Specific sequences in a sample of nucleic acid.

Who invented the Southern blot technique?

Sir Edwin Southern.

What is meant by 'denaturation' in nucleic acid techniques?

The process of separating the double-stranded DNA into single strands.

What are restriction enzymes used for in a Southern blot?

They cut DNA at specific sequences.

What is the purpose of gel electrophoresis in Southern blotting?

To separate DNA fragments by size.

What is a fluorophore?

A label that allows for the detection of a probe.

What is the main radiation hazard associated with P32?

Internal and external exposure can result from their penetration ability.

What does Southern blotting require after the DNA is transferred to the membrane?

Rinsing the membrane with buffer.

What is the purpose of stringency washes in probe hybridization?

To remove unbound probes and increase specificity.

Which radioactive isotope is commonly used in nucleic acid probes?

32P (Phosphorus-32).

How is a Northern blot different from a Southern blot?

A Northern blot involves RNA rather than DNA and does not require digestion.

What is FISH used for?

To identify chromosomal abnormalities.

What is the resolution range for FISH techniques?

1-5 kb.

What types of cells can be used for FISH?

Cultured cells and FFPE (formalin-fixed paraffin-embedded) tissue.

What is the role of DAPI in FISH?

A counterstain that binds to DNA and fluoresces under UV light.

What is the function of a covering slip in FISH?

To keep the probe in place during hybridization.

What happens during the hybridization step in FISH?

Probes are allowed to bind to their complementary DNA sequences overnight.

How is DNA microarray technology primarily used?

For the analysis of gene expression patterns.

What is cDNA microarray used for?

To analyze gene expression by comparing mRNA from different cell populations.

What does higher intensity of red or green signify in microarray analysis?

Red indicates more patient DNA; green indicates less patient DNA.

What type of probes does comparative genomic hybridization use?

Probes that cover the entire genome.

What are the two fluorophores used in aCGH?

Cy3 for patient DNA and Cy5 for control DNA.

How should the samples be labeled for microarray analysis?

One with a red fluorophore and the other with a green fluorophore.

What are the advantages of FISH?

Quick, high resolution, can be done on uncultured cells, and can confirm g-banding results.

What are the disadvantages of FISH?

Expensive, limited information, and potential technical problems.

What does the Southern blot technique primarily analyze?

Specific DNA sequences in a genome.

What is the process of hybridization?

The binding of a probe to its complementary DNA sequence.

What safety precautions should be considered when using radioactive probes?

Risks of external and internal exposure to radiation.

What medium is used to run a Southern blot gel?

A gel typically made of agarose or polyacrylamide.

What role does sodium hydroxide (NaOH) play in the Southern blot protocol?

It is used to denature the double-stranded DNA.

How is the efficiency of DNA transfer to a membrane checked?

By staining the gel and ensuring it shows no DNA after transfer.

What are some blocking agents used in Southern blotting?

Denatured salmon sperm DNA, SDS, Denhardt’s reagent.

What is the purpose of formamide in hybridization solutions?

To reduce the melting temperature of DNA and control hybridization.

What should be done if there is unbound probe after the hybridization step?

Wash the membrane with stringency washes.

What visual method is used to check for presence of the 32P labeled probes?

Autoradiography.

What is a 'unique sequence probe'?

A probe created with cloning or PCR that targets a specific sequence.

What is the significance of the elastin (ELN) gene in Williams syndrome?

Haploinsufficiency of this gene causes supravalvular aortic stenosis (SVAS).

What is karyotyping?

The process of arranging chromosomes into a karyotype to examine them for abnormalities.

What characteristic features are associated with Williams syndrome?

Distinctive facial appearance, cardiovascular problems, and cognitive disabilities.

Why is comparative genomic hybridization useful?

To detect copy number alterations between two samples.

What is the recommended temperature for probe hybridization in FISH?

Initialized at 75 °C to denature then lowered to 37 °C overnight.

What type of cytogenetic abnormalities can FISH detect?

Deletions, amplifications, and chromosomal translocations.

What must be done before applying FISH probes to slides?

The cells must be fixed and pre-treated.

What is the main purpose of using DNA microarrays in research?

To screen for gene expression differences in various samples.