TMC 6 Flashcards
List the ways that DNA can be denatured.
Heat
High pH
Force
Specific enzymes
Name the enzyme that unwinds DNA
DNA helicase
What is the melting temperature for a G:C bp?
4C
What is the melting temperature for a A:T bp?
2C
At what temperature does all DNA become single stranded?
95C
What is meant by DNA melting temperature?
Temperature required to separate two strands of DNA by breaking the hydrogen bonds that bind the nucleotides together
What is the definition of DNA hybridisation/annealing?
When two single strands of complementary DNA are free in a solution the strands will bind to each other in their complementary areas as long as the conditions are suitable for such binding e.g. correct temperature
Which chromosome is the cystic fibrosis transmembrane regulator gene found on?
Chromosome 7
What is the role of the CFTR protein?
Chloride absorption and secretion
What is the most common cystic fibrosis mutation?
Delta F508 - Deletion of F at position 508 due to a deletion of CTT in the gene
Explain how DNA probes and hybridization can be used to detect the normal and the commonly mutated allele F508 on the CFTR gene in a DNA sample from an individual.
- Blood sample- nitrocellulose.
- DNA probes to detect the phenylalanine at position 508 – ctt, gaa.
- Temp 95
- Reduced to just below the temp of the probe.
- Given time to hybridise.
- The membrane is removed and washed with more solution at the probe hybridisation temp. Removes any unbound probe.
- The membrane is scanned using a fluorescent scanner. If the probe has bound to the DNA it means that the complementary bases for the phenylalanine must be present ctt, gaa If not then the gene must be absent.
Explain how a primer can be extended on a DNA template, draw an example to illustrate your answer ensuring that you show direction.
5’ to 3’ direction
dNTPs must be present and added to the 3’ free end of the primer
The DNA polymerase catalyses the formation of the 5’ to 3’ phosphodiester bonds.
Uses the DNA strand as a template
Explain the process of PCR
PCR is a method inn which certain fragments of DNA are amplified
In order to amplify a specific sequence you must identify 2 RNA primers that will flank the segment of DNA you wish to amplify
Heat the DNA to 94 degrees
Melts the hydrogen bonds and separates the DNA into 2 separate strands
Place excess forward and reverse primers into the liquid and allow them to anneal to DNA by reducing it to a temperature under the melting temperature of the primers.
dNTPs, DNA pol - TAQ, Mg++ buffer allows DNA synthesis to occur
Allows for hybridised primers to be extended
Explain the process of gel electrophoresis.
DNA negatively charged
Positive electrode
Agarose
Positive charge pulls the DNA
Smaller fragments move more quickly - migration rate directly related to DNA’s size
Fluorescent dye such as ethidium bromide
Explain qPCR
The number of cycles of PCR required to
generate a set amount of amplified DNA
from a sample is dependent on the number of target molecules in the
sample.
The more target molecules present the lower the number of PCR cycles required to generate the set amount of amplified DNA.
Thus PCR can be used to measure the relative amount of target molecules between samples
Used extensively for measuring virus quantity in infections and mRNA produced from genes
