Descrimination techniques Flashcards
What are the three general categories of discrimination techniques?
- Electrophoretic separation
- Alternatives to electrophoresis
- Hybridization assays
What is the purpose of electrophoretic separation?
To physically separate nucleic acids based on molecular weight and shape.
What is the purpose of hybridization assays?
To visualize specific nucleic acids using probes.
Why are DNA and RNA negatively charged?
Due to the phosphate groups in their backbone.
What is the direction of nucleic acid migration during electrophoresis?
Towards the anode (positive electrode).
How are DNA fragments visualized after electrophoresis?
Using a dye (e.g., ethidium bromide) that binds to DNA and fluoresces under UV light.
What are the two types of polymers used in electrophoresis?
Agarose and polyacrylamide.
What size range of nucleic acids can agarose gels separate?
From 20 bp to over 10 Mb.
What is the resolution limit of agarose gels?
A size difference of 2% to 5%.
How are agarose gel results recorded?
By taking a photographic image of the stained gel under UV light.
What is polyacrylamide gel primarily used for?
High-resolution separation of short nucleic acid molecules (up to 1 kb).
What is a key application of polyacrylamide gels?
DNA sequencing studies.
How are polyacrylamide gels stained?
Using fluorescent stains or silver staining.
What factors affect the migration of nucleic acids during electrophoresis?
- Size and complexity of nucleic acids
- Concentration of ethidium bromide
- Gel concentration
- Applied electric field
How does DNA conformation affect migration?
Supercoiled DNA moves faster than relaxed DNA.
How does gel concentration affect DNA separation?
Higher gel concentration reduces pore size, improving separation of smaller DNA molecules.
What is the effect of increasing voltage during electrophoresis?
DNA migrates faster.
Who developed the Southern blot technique?
Edward Southern in 1975.
What is the purpose of Southern blotting?
To detect specific DNA fragments in a DNA sample.
What are the general steps of Southern blotting?
- Extract and purify DNA
- Restrict DNA with enzymes
- Denature and electrophorese DNA
- Transfer to nitrocellulose paper
- Incubate with a single-stranded DNA probe
- Wash off unbound probes
- Autoradiograph
What is the main difference between Southern and Northern blotting?
Northern blotting uses RNA instead of DNA.
What is the purpose of Northern blotting?
To observe gene expression patterns in different tissues, organs, or conditions.
How is RNA visualized in Northern blotting?
Using a single-stranded DNA probe that binds to complementary RNA sequences.
What are the applications of Northern blotting?
- Observing gene expression in normal vs. diseased conditions
- Detecting overexpression of oncogenes or downregulation of tumor-suppressor genes
- Monitoring gene expression in organ transplant rejection
What are the two main DNA sequencing techniques?
Sanger dideoxy DNA sequencing and Pyrosequencing.
What is the principle of Sanger sequencing?
Single-stranded DNA molecules differing by one nucleotide can be separated by polyacrylamide gel electrophoresis.
What is the role of dideoxynucleotides (ddNTPs) in Sanger sequencing?
They terminate DNA synthesis because they lack a 3’-hydroxyl group.
How is DNA sequence determined in automated Sanger sequencing?
Each ddNTP is tagged with a fluorescent marker, and a laser detects the emitted light as DNA fragments pass through a detector.
What is the main advantage of pyrosequencing?
It allows DNA sequencing without electrophoresis.
What enzymes are used in pyrosequencing?
DNA polymerase, ATP sulfurylase, luciferase, and apyrase.
How is light produced in pyrosequencing?
Pyrophosphate released during nucleotide incorporation is converted to ATP, which drives the conversion of luciferin to oxyluciferin, producing light.
What is HPLC used for in molecular diagnostics?
To separate and purify oligonucleotides.
What type of chromatography is used in HPLC for nucleic acids?
Ion-pair, reversed-phase chromatography.
How is mass spectroscopy used in molecular diagnostics?
To detect polymorphisms by measuring the mass difference between alleles.
What is the advantage of mass spectroscopy over other techniques?
It does not require labels because alleles differ in mass.
Why is ethidium bromide used in gel electrophoresis?
It intercalates into DNA and fluoresces under UV light, allowing visualization of DNA bands.
What is the difference between agarose and polyacrylamide gels?
Agarose gels are used for larger DNA fragments (20 bp to 10 Mb), while polyacrylamide gels are used for high-resolution separation of smaller fragments (up to 1 kb).
How does pyrosequencing differ from Sanger sequencing?
Pyrosequencing measures light produced during nucleotide incorporation, while Sanger sequencing relies on chain termination and electrophoresis.
