6. Probe Based Nucleic Acid Techniques

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 type of labels can probes contain?

Probes can contain labels such as fluorophores or radioactive isotopes like P32.

What is the purpose of a Southern blot?

A Southern blot is used to detect specific sequences in a sample of nucleic acid.

What enzyme is commonly used in the preparation of DNA for a Southern blot?

Restriction enzymes are used to cut DNA at specific sequences.

How are DNA fragments separated in Southern blotting?

DNA fragments are separated using gel electrophoresis.

What needs to be done to DNA before transferring it to a membrane in a Southern blot?

The dsDNA must be denatured.

What is the next step after loading DNA samples in a Southern blot?

After loading, the gel is run to achieve electrophoresis.

What type of membrane is commonly used to transfer DNA in Southern blotting?

A nylon membrane is commonly used.

What is the significance of probe labeling in Southern blots?

Labels (fluorescent or radioactive) allow for detection of specific DNA fragments that have hybridized with the probe.

What is one hazard associated with using radioactive probes like P32?

P32 presents external and internal exposure hazards due to its radiation.

What is fluorescence in situ hybridization (FISH) used for?

FISH is used to identify chromosomal abnormalities and specific types of RNA in cells.

How is RNA prepared for Northern blotting?

RNA does not need to be digested and must be denatured to prevent secondary structures.

What can FISH detect?

FISH can detect deletions, amplifications, translocations, and other chromosomal abnormalities.

What is the main advantage of using FISH compared to other chromosomal techniques?

FISH provides high resolution and specificity for detecting chromosomal abnormalities.

What does high stringency in washes during FISH indicate?

High stringency means fewer unbound probes remain after the wash, which indicates stronger binding.

What common counterstain is used in FISH analysis?

DAPI is often used as a counterstain in FISH analysis.

What is a karyotype?

A karyotype is a visual representation of an individual's complete set of chromosomes.

What process is used to visualize DNA in Southern blots?

Autoradiography or fluorescence imaging is used to visualize the DNA.

What is the function of the hybridization buffer in DNA microarray?

The hybridization buffer stabilizes DNA binding and controls conditions for hybridization.

In cDNA microarrays, what is the primary purpose of reverse transcription?

Reverse transcription converts mRNA into cDNA for analysis.

How does microarray technology distinguish between control and patient DNA?

By using two different fluorophores to label control (green) and patient (red) DNA.

What happens if more patient DNA binds to a probe in a microarray?

If more patient DNA binds, the spot appears red, indicating overexpression.

What are some applications for DNA microarrays?

Applications include gene expression analysis, detection of SNPs, and comparative genomic hybridization.

How is Williams Syndrome identified at the genetic level?

Williams Syndrome is identified as a microdeletion syndrome on chromosome 7.

What type of anomalies can DNA microarrays help detect?

Microarray can help detect copy number alterations, aneuploidies, and specific genetic disorders.

What is the result of hybridization in FISH?

The process results in specific binding of labeled probes to target DNA regions.

Why is tape used over coverslips in FISH?

Tape is used to seal coverslips and keep them in place during analysis.

What is the importance of normalizing DNA in Southern blotting?

Normalization ensures equal intensity bands on the gel for accurate comparison.

How is DNA transferred from gel to membrane in Southern blotting?

Through capillary action using a buffer solution.

What is one disadvantage of the FISH technique?

FISH can be expensive and may have technical problems.

What role do blocking agents play in FISH?

Blocking agents prevent non-specific binding of probes during hybridization.

What is required to confirm the efficiency of DNA transfer in Southern blots?

Staining the gel to check for remaining color indicates successful transfer.

What is a common use for comparative genomic hybridization (CGH)?

To detect copy number alterations between clinical samples.

What is the common outcome of overexpression detected by microarrays?

Overexpression typically indicates duplication or amplification of genes.

During FISH preparation, why is colcemid used?

Colcemid is used to arrest cells in metaphase for easier chromosome visualization.

What can be inferred by visualizing a karyotype?

Karyotype analysis can reveal abnormalities like deletions, duplications, and translocations.

How can different fluorophores help in microarray analysis?

Different fluorophores can indicate varying levels of gene expression between samples.

What is the purpose of normalization in DNA microarrays?

Normalization corrects for variance in fluorescence intensity for accurate data interpretation.

What is meant by the term 'stringency' in FISH washes?

Stringency refers to the conditions (temperature/salt concentration) that determine the specificity of probe binding.

How is a DNA microarray arranged?

DNA sequences are immobilized onto precise spots on a solid support like a glass slide.

What must be done to the slides before applying probes in FISH?

Slides must be treated and aged to prepare for probe application.

What is a frequent reason for using FISH in clinical settings?

To confirm suspicions of chromosomal abnormalities found through other methods.

What does a positive control in FISH help demonstrate?

It shows that the probe is functioning properly and binding as intended.

Which method is often used to separate the bands following G-banding?

Giemsa staining is used after treating chromosomes with trypsin.

What is the genetic hallmark of Williams Syndrome?

It is characterized by a microdeletion on chromosome 7, specifically at 7q11.23.