Advanced molecular techniques Flashcards
What are allele-specific primers?
Refers to primers whose 3’ base sequence is complementary to one allele but not the other
What happens if a primer’s 3’ end is complementary to a DNA sequence in PCR?
Taq polymerase can elongate new DNA strand
What happens if a primer’s 3’ end is not complementary to a DNA sequence in PCR?
Taq polymerase cannot elongate the new DNA strand
What are allele-specific probes?
Refers to probes which are completely complementary to base sequence of one allele but not the other
How do allele-specific probes bind to different alleles?
Bind tightly to alleles they are specific to
Bing less tightly to other alleles
How would results of Southern blotting using allele-specific probes be interpreted?
Darker band means more allele-specific probe bound
so allele specific to probe is present there
What is the first step in reverse transcriptase PCR?
Extract mRNA for wanted gene from the cell
What is done to the extracted mRNA in reverse transcriptase PCR?
Add DNA primer to it
which contains lots of T bases
What does the DNA primer do with mRNA in reverse transcriptase PCR?
DNA primer containing lots of T bases
binds to polyA tail in mRNA
What does reverse transcriptase do the the DNA primer bound to mRNA in reverse transcriptase PCR?
Reverse transcriptase elongates the DNA primer as cDNA
by reading the mRNA
What happens to mRNA after the formation of cDNA in reverse transcriptase PCR?
Digested by RNase enzyme
What happens to the single-stranded cDNA in reverse-transcriptase PCR?
Undergoes one cycle of PCR
one DNA primer binds
to produce double-stranded cDNA
What happens to double-stranded cDNA in reverse-transcriptase PCR?
Undergoes many cycles of PCR
both DNA primers bind
to produce copies of cDNA
What does a microarray chip look like?
Slide with grid on it
What does each dot on a microarray chip contain?
Single-stranded sections of DNA from one gene only
What does microarray conditional gene expression compare?
Compares gene expression of a gene
in normal cell to diseased cells
What is the first stage of microarray conditional gene expression?
Reverse transcriptase PCR for normal cell and diseased cell
to produce single-stranded cDNA
How is cDNA from the normal cell and diseased cell labelled in microarray conditional gene expression?
Normal cell cDNA is fluorescently labelled green
Diseased cell DNA is fluorescently labelled red
What is done to the normal cell cDNA and diseased cell cDNA in microarray conditional gene expression?
Add the cDNA to the relevant gene in the microarray chip
How are the results of microarray conditional gene expression interpreted?
Look at colour of dot
- if more green means diseased cell has reduced gene expression for that gene
- if more red means diseases cell has increased gene expression for that gene
- if yellow means equal expression in each cell
What does array comparative genome hybdridisation compare?
Compares genes in normal cell to genes in diseased cell
What is the first stage of aCGH?
Extract all DNA from normal cell and from diseased cell
What is done to the DNA extracted from the normal cell and the diseased cell in aCGH?
Digest with restriction enzymes
Fluorescently labelled
- normal cell is green
- diseased cell is red
What is done to the fluorescently labelled DNA fragments in aCGH?
Added to the microarray chip
