MCQ2 Prep Flashcards
Restriction Fragment Length Polymorphism (RFLP)
A technique used to analyze variations in DNA sequences by cutting and separating DNA fragments and using gel electrophoresis.
Restriction enzymes (endonuclease)
A protein that recognizes a specific palindromic DNA sequences and cuts the DNA at this site.
Restriction patterns
The unique arrangement of DNA fragments produced after digestion with a restriction enzyme, which can be analyzed using gel electrophoresis to detect mutations or polymorphisms.
Genomic DNA
A complete set of DNA within an organism including both coding genes and non-coding genes.
Gel electrophoresis
A technique used to separate DNA fragments by size using an electric field. Smaller fragments migrate faster through the gel matrix, allowing visualization of restriction fragment patterns.
How do mutations affect restriction sites?
Mutations can create or destroy restriction sites, altering the fragment pattern in the RFLP analysis.
What is EcoR1?
A restriction enzyme that recognizes the sequence GAATTC and cuts between G and A, generating sticky ends.
Sticky vs blunt ends in restriction digestion.
Sticky ends have overhanging DNA fragments, blunt ends have no overhangs.
DNA ligase
An enzyme that joins DNA fragments by sealing phosphodiester bonds between adjacent nucleotides.
Polymerase Chain Reaction
Technique used to amplify specific DNA sequences.
Taq polymerase
From Thermus aquaticus, this DNA polymerase withstands high temperatures, ideal for PCRs.
Primers in PCR
Single stranded DNA sequences that bind in complementary regions to the template DNA to initiate DNA synthesis.
Thermocycler
A laboratory device used in PCR that rapidly changes temperature to allow denaturation, annealing and extension to occur in controlled manner.
Palindromic sequence
Short DNA sequence that is read the same forward and backward on complementary strands.
How does PCR detect genetic mutations?
Primers designed for specific mutations allow PCR amplification only if the mutation is present.
How is PCR combined with RFLP analysis?
PCR amplifies a target DNA region, restriction enzymes digest PCR product, Gel electrophoresis visualizes the fragment patterns, revealing mutations or polymorphisms.
Steps of PCR
- Initial Denaturation – 95°C, 3 min → separates DNA
- Denaturation – 95°C, 30 sec → single strands form.
- Annealing – 55°C, 30 sec → primers bind to target sequences.
- Extension – 72°C, 1 min/kb → Taq polymerase synthesizes new DNA.
- Final Extension – 72°C,10 min → Completes any unfinished strands.
- Hold – 4°C, indefinite → Stores PCR product for analysis.
