Molecular Biological Techniques

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What is the basic structure of a DNA molecule?	Phosphate backbone, a pentose sugar (deoxyribose) and a nitrogenous base
What is the difference between purines and pyrimidines?	Purines contain 2 rings whereas pyrimidines contain only 1
What is the general function of restriction enzymes?	They hydrolyse phosphodiester bonds
What do type II restriction enzymes do? (2)	1. Recognise a specific sequence of DNA 2. Cut that sequence
Why are the DNA sequences targeted by restriction enzymes usually palindromic?	They often work as homodimers
What are the 2 possible outcomes of the hydrolysis of phosphodiester bonds by restriction enzymes?	1. Blunt ends 2. 'Overhangs' which are therefore able to re-anneal
Which enzyme can form new phosphodiester bonds?	DNA ligase
Which biological technique can be used to separate DNA molecules?	Gel electrophoresis
Describe how gel electrophoresis works	Samples of DNA are placed in wells at one end of a gel, including a standard sample. A cathode is placed at the end of the gel with samples, and an anode at the other end. Since DNA is negatively charged, the samples will move towards the anode. The size of the DNA samples will determine how far along the gel they move, and so can be measured against the standard sample.
What is restriction mapping used for?	A way of finding out where the restriction sites are in an unknown molecule, particularly useful in describing plasmids
How does restriction mapping work?	Create a sample of DNA + restriction enzyme 1, a sample of DNA + restriction enzyme 2 and a sample of DNA + both restriction enzymes. Perform gel electrophoresis on the samples, to find the restriction sites, as you will be able to see how many base pairs long each fragment is
Which technique is used to amplify known fragments of DNA?	PCR
What are the 3 basic stages of PCR?	1. Denaturation 2. Annealing 3. Extension
What components are needed for PCR? (5)	1. DNA template 2. Primers 3. Free nucleotides 4. DNA polymerase 5. buffer solution
What happens during the denaturation stage?	DNA strands are forced apart by applying heat of 95 degrees celcius
What happens during the annealing stage?	Temperature is reduced to 50-65 degrees celcius and primers bond to the DNA with hydrogen bonds
What happens during the extension phase?	At 72 degrees celcius, DNA polymerase binds to the primers and forms a complimentary strand using free nucelotides.
Why is the DNA polymerase Taq used?	Taq comes from bacteria that live in hot springs, so can survive in high temperatures such as those experienced in the denaturation stage
In which circumstances is PCR useful?	For strands of DNA <100 bp long, and if you already know the sequence of that DNA
What could be a clinical application of these techniques?	Testing for sickle cell - perform PCR on a section of the patients DNA, then use a restriction enzyme that will only work on a normal beta-globin gene. Do gel electrophoresis on the products to observe if the DNA has been cut by the restriction enzyme or not. If not at all = sickle cell anaemia. If cut slightly but not totally = carrier
Describe the process of cloning to produce human insulin. (6 stages)	1. Isolate the section of DNA that codes for insulin, using restriction enzymes 2. Insert the DNA into a plasmid vector using ligase 3. Introduce the plasmid into a bacterium (E.coli) using electroporation 4. Culture the bacteria 5. Induce expression of the protein so it produces insulin 6. Purify the product
Why would you perform a chromosome analysis?	To detect major syndromes
What could be detected on a chromosome analysis? (3)	1. Aneuploidies 2. Large deletions/duplications 3. Translocations between chromosomes
What is G-banding?	Staining of chromosomes with a Giesma stain while they are at metaphase, to look for any extra or missing choromosomes or large translocations/deletions/duplications
What is the biggest disadvantage of G-banding?	Takes several days
What is FISH?	Fluorescent in-situ hybridisation, uses fluorescent markers to see if specific parts of the genome are abnormal (translocated, duplicated or deleted)
How does FISH work?	Add a fluorescent marker for the chromosome you are interested in, and also a fluorescent marker for the specific DNA sequence on that chromosome you want to study. This will identify if the gene is present or translocated.
What are the disadvantages of FISH?	1. Takes several days 2. You need to know what you are looking for
What is QF-PCR?	Quantitative fluorescence polymerase chain reaction
What does QF-PCR achieve?	Detects aneuploidies (abnormal numbers of chromosomes) by measuring the number of repetitive units found (microsatellites)
What are microsatellites?	Short repeats of DNA sequence found across the entire genome
How is QF-PCR better than FISH or G-staining?	Takes only 24-48 hours
What is aCGH?	Array - comparative genomic hybridisation
What does aCGH detect?	Deletions and duplications
Describe the process of aCGH	Metaphase chromosomes are extracted from a control and also the patient. Each is given a different fluorescent marker, then mixed and denatured to form a hybrid DNA. A computer measures the fluorescence intensity from each of the DNA samples in the hybrid, giving a ratio between them. If the ratio is more than 1 for a particular chromosome, then there is a duplication in that chromosome. Conversely, if the ratio is less than 1 for a chromosome, there is a deletion in that chromosome.
What is Sanger sequencing?	A way of studying the individual bases within a gene to look for substitutions
How can you 'paint' a specific chromosome so it can be identified by FISH?	Prepare a metaphase spread of chromosomes, then isolate the desired chromosome. Amplify that chromosome and add a tag (dye). This chromosome can then be hybridised into a sample, and will appear with the tag.

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