Lecture 4 Part 1 Flashcards
What are DNA restriction and sequencing?
Fundamental techniques in molecular biology used for precise cutting of DNA and determining nucleotide order.
What are restriction enzymes?
Proteins that recognize specific DNA sequences and cut at or near these sites.
Where do restriction enzymes naturally occur?
In bacteria as a defense mechanism against foreign DNA.
What factors affect the activity of restriction enzymes?
Temperature, presence of cofactors, correct pH, and salt conditions.
What is a palindromic sequence in the context of restriction enzymes?
A sequence that reads the same forward and backward on complementary strands.
What specific sequence does EcoRI recognize?
GAATTC.
What type of ends does EcoRI produce after cutting?
Sticky ends.
What is the lottery effect in relation to recognition sites?
The longer the recognition site, the less frequent it will be in a random genome.
What percentage of DNA sequences are the same among individuals of a species?
90%.
What are polymorphisms?
Individual genetic differences that include differences in restriction enzyme cleavage sites.
How can genetic traits be used for identification?
Each variant of polymorphism can be inherited, allowing for personal or parental identification.
What are the types of restriction enzymes?
Type I: Cuts at random locations far from recognition sites.
Type II: Cuts at specific recognition sequences.
Type III: Cuts a short distance from the recognition site.
Type IV: Targets modified DNA.
What is one application of restriction enzymes in genetic engineering?
To insert or remove genes.
What do restriction fragment length polymorphisms (RFLPs) allow for?
Forensic analysis.
What is the first step in the restriction digestion process?
Mixing DNA sample with restriction enzyme and buffer containing Mg²⁺.
At what temperature are many restriction enzymes incubated for optimal activity?
37°C.
What principle does gel electrophoresis rely on?
DNA molecules are negatively charged, allowing migration in an electric field.
What happens to DNA samples during gel electrophoresis?
They are loaded into wells of an agarose gel and migrate toward the positive electrode.
How do smaller DNA fragments behave in gel electrophoresis compared to larger fragments?
Smaller fragments migrate faster.
What is used to visualize DNA in gel electrophoresis?
Ethidium bromide or other dyes under UV light.
What is Southern blotting?
A method used to detect specific DNA sequences in a sample.
What are the main steps involved in Southern blotting?
Transferring DNA from gel onto a membrane.
Hybridizing with a labeled probe.
Detecting hybridized fragments.
What does DNA sequencing determine?
The order of nucleotides (A, T, C, G) in a DNA strand.
Who developed the Sanger sequencing method?
Frederick Sanger.
What type of technique is the Sanger sequencing method?
Chain termination technique.
What is the role of template DNA in Sanger sequencing?
The DNA strand to be sequenced.
What is a primer in the context of DNA sequencing?
A short DNA sequence that initiates DNA synthesis.
What enzyme is responsible for adding nucleotides in Sanger sequencing?
DNA polymerase.
What are dNTPs?
Normal building blocks of DNA (A, T, C, G).
What are ddNTPs and their function in Sanger sequencing?
Modified nucleotides that terminate chain elongation.
Fill in the blank: The Sanger sequencing method uses _ that lack hydroxyl -OH preventing polymerisation reaction.
ddNTPs.
What is the first step in Sanger sequencing?
DNA is denatured into single strands.
What happens after primers bind to the template DNA in Sanger sequencing?
DNA polymerase extends the new strand by adding normal nucleotides (dNTPs).
What results from incorporating a small amount of ddNTP during Sanger sequencing?
Stopping elongation at a specific base.
How are DNA fragments separated in Sanger sequencing?
Capillary electrophoresis.
What does a chromatogram display in Sanger sequencing?
Coloured peaks corresponding to the detected nucleotides.
True or False: Sanger sequencing processes multiple DNA strands simultaneously.
False.
What is a key advantage of Next-Generation Sequencing (NGS)?
Higher throughput (millions of sequences at once).
Name one application of DNA sequencing.
Genetic testing, forensic science, evolutionary biology, biotechnology.
What role do magnesium ions (Mg²⁺) play in PCR and DNA reactions?
Essential cofactor for DNA polymerase and restriction enzymes.
Fill in the blank: In PCR, Mg²⁺ concentration affects _.
Enzyme activity and primer annealing.
How does too little magnesium ions (Mg²⁺) affect PCR?
Reduces efficiency.
How does too much magnesium ions (Mg²⁺) affect PCR?
Leads to non-specific binding.
