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
How are the results of aCGH interpreted?
Look at colour of dot for each gene
- if more green, means diseased cell has deletions
- if more red, means diseased cell has duplications
- if yellow, means normal
Are minisatellites in coding DNA or non-coding?
Non-coding
What are minisatellites?
Repeating base sequences
How specific are minisatellites to an individual? Why?
Unique to individuals
but somewhat resemble minisatellites of parents, since inherited DNA from parents
What are the processes involved in DNA fingerprinting?
Restriction analysis
followed by Southern blotting
What are the probes complementary to in DNA fingerprinting?
Complementary to minisatellites in DNA
How are the results of DNA fingerprinting interpreted?
Compare banding patterns
- somewhat similar between parent and child
- exactly similar if of same person
What are the uses of DNA fingerprinting?
Forensics - suspects to site of crime
Paternity testing
What is the first stage in Fluorescent In Situ Hybridisation (FISH)?
Add fluorescently labelled probes to DNA
What do the fluorescently labelled probes in FISH do?
Bind to complementary base sequences in DNA
How are the results of FISH interpreted?
Look at fluorescently labelled probe under microscope
where it had bound to DNA
What is the use of FISH?
Detects mutations
What is the first stage of whole chromosome pointing?
Add fluorescently labelled probes to all of chromosome length
How are the results of whole chromosome painting interpreted?
Look at colour of entire chromosome
What are the uses of whole chromosome painting?
Detect numerical abnormalities
Detect structural abnormalities
How are the results of karyotyping interpreted?
Look at banding pattern of chromosomes formed by staining
