Flashcards in Molecular Diagnosis Deck (39):
How is our own DNA protected from restriction enzymes?
How can we isolate particular pieces of DNA from a sequence?
Use multiple restriction enzymes.
What is DNA electrophoresis?
It is a method which can be used for visualising colourless DNA. Fragments are separated using a negative charge. Fragments have to move on the plate so larger molecules move less far, gradient of molecule sizes created.
What are the four requirements for gel electrophoresis?
Gel - allows separation, Buffer - maintains charge, Power supply - creates charge difference, stain - visualise DNA.
What is the function of DNA ligase in molecular diagnosis?
When complementary base sequences align, this can lead to the formation of new bonds.
What is used in gene cloning?
Plasmids from Bacteria are used in gene cloning.
What is recombinant DNA?
This is the plasmid ring with the DNA in it as well.
What is the use of cloning genes?
We can use cloned genes in order to make useful proteins such as insulin.
What is genetic screening?
This is where we are looking for a specific gene. To look at this properly we clone genes.
What is PCR?
Polymerise chain reaction. It is a method for obtaining large amounts of a particular piece of DNA for analysis.
What is a primer?
This is a short molecule which is complementary to part of the DNA sequence. It is a site where DNA replication begins.
Explain the cycle process of PCR.
First DNA is heated to 95 degree so that it is denatured. It is then cooled to 60 so that primers anneal and then heated to 75 when replication takes place. The DNA is then reheated.
What is used so that DNA polymerase denaturing is not a problem in PCR?
Taq, thermostable DNA polymerase so that it does not have to be replaced with each cycle.
What is the use of PCR?
PCR allows amplification of a specific piece of DNA which can then be investigated for base mutations or deletions/insertions.
State the difference between protein and DNA gel electrophoresis.
Protein gel electrophoresis is done vertically rather than horizontally so proteins move down the plate.
What is Isoelectric focusing in protein gel electrophoresis?
This is where proteins are separated on the basis of their charge. When they reach the pH = pI they cease to have a charge and so they do not move as they are not attracted to either dipole.
How are proteins separated in SDS-Page?
In this process all proteins are denatured and given a uniform negative charge. They are then separated by size as larger proteins will move more slowly across the gel.
What is 2D PAGE?
This is where proteins are initially separated by Isoelectric focusing and then they are separated by size.
What methods can we use to identify proteins after they have been separated?
Digest with trypsin and perform mass spectrometry, enzymatic cleavage, chemical cleavage or antibodies which will recognise a few amino acids on protein.
What is the difference between polyclonal and monoclonal antibodies?
Polyclonal antibodies are antibodies which can detect one antigen but there are multiple different antibodies which recognise different epitopes. Monoclonal antibodies are specific to one antigen but there is one antibody and one epitope.
What is a restriction enzyme?
This is a specific endonuclease which cuts a specific DNA sequence, usually a palindrome.
What is western blotting?
This is where there is a nitrocellulose replica of the glee electrophoregram which is incubated with primary antibodies and then and enzyme linked to a second antibody is used to detect this binding.
What is an Enzyme-linked immunoabsorbent assay?
This is where there is an antigen coated well to which a specific enzyme linked antibody can bind. When substrate binds it is converted to a coloured product by the enzyme, therefore indicating how much antibody was bound.
What is a radioimmunoassay?
This is the same as an ELISA however it uses a radio labelled primary antibody.
Why is measurement of enzymes an important diagnostic technique?
We can detect: metabolic disorders of tissues, and also we can diagnose disease.
There are many enzymes which when they become present in the blood indicate disease.
How can enzymes be used to measure clinically important metabolites?
On test strips, enzymes can convert these metabolites to product. This product can then be detected and measured.
What is DNA sequencing?
This is a process by which we can find out the genomic code and look to see if there are mutations and insertions or deletions.
Describe briefly the process of DNA sequencing.
Dideoxynucleotides are added to the mixture rather than just deoxy ones. This means that in replication, there is a chance that each time it is a specific base the dideoxy form will take the space. This means that the sequence stops. By running it the fragments are separated and we can see the order of the bases.
What is DNA hybridisation?
This is where we can denature DNA and then insert a radioactively labelled DNA probe into the mixture. This allows us to identify fragments of DNA.
What is the difference between southern blotting and northern blotting.
Both are processes which involve using DNA probes to find complementary base sequences after gel electrophoresis. Southern uses DNA to find DNA, Northern uses DNA to find RNA.
Why after gel electrophoresis is there transfer to a nylon membrane in southern blotting?
DNA needs to be transferred to a solid support. this can be done by either capillary action or it can also be done by using a voltage to move the negatively charged DNA from the gel to filter.
Once DNA is on the filter, what happens in southern blotting?
The filter is placed in a solution with labelled DNA probes. These will bind to complementary base sequence. The filter is then washed before the probes are visualised.
Explain the properties of probes in southern blotting.
Probes do not have to be 100% complementary because they will still bind just not as tightly. They also do not have to completely align as long as overlap is sufficient. Probe position will not affect the position of the band identified.
What is an allele specific primer in PCR?
This will only produce a PCR product when the correct sequence is present - allele specific.
How can we analyse RNA?
RNA is an unstable molecule so first it is converted to DNA by RNA reverse transcriptase. In order for this we simply need a T primer which can bind to the polyA tail.
What is a microarray?
This is where dots of either red, green or brown can be seen. mRNA from two cells is used and then fluorescent probes are used. This allows us to see where there is missing RNA and in which cell this occurs.
What is an important process in DNA fingerprinting?
PCR. The amount of DNA we have is very limited, so it must be amplified by PCR.
What is Karyotyping?
This is looking at banding patterns on Chromosomes.