Term
| what is Agarose gel made from? |
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Definition
Long polysacharide made of modified galactose units.
it is INERT, has No charge and comes as Powder |
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Term
| what does Agarose gel electrophresis (AGE) do to DNA? |
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Definition
| it seperates DNA fragments by size |
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Term
| what overall charge does DNA have? |
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Definition
| a negative charge thus it AGE it flows towards the positive charge |
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Term
| in AGE, why is important to include glycerol or sucrose buffer with the DNA sample? |
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Definition
| to increase the density so the solution will sink in the gel and not diffuse out |
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Term
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Definition
| where a brief electrical pulse is applied to a solution containing cells. the pulse creates temporary holes in the plasma membrane through which DNA can enter |
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Term
| ethidium bromide-stained gel is viewed under UV light to see the markings of DNA from agarose gel electrophoresis but why is it not used anymore? what colour does it show under UV light? |
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Definition
it is mutagenic, damages DNA and also a carcinogen
(GelRed is a safer alternative)
under UV light ethidium bromide is fluorescent pink |
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Term
| the normal allele and sickle cell allele have different fragment lengths. which procedure can help to distinguish this? |
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Definition
RFLP
restriction fragment length polymorphism. this is when you carry out electrophoresis of normal and sickle-cell alleles |
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Term
| from just carrying out RFLP you cannot distiguish the DNA. why is this? |
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Definition
| because digested DNA appears as a smear. you need to further process this with a technique known as Southern Blotting |
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Term
| in southern blotting the probe is usually a radioactive or relevantly labled .......... molecule |
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Definition
|
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Term
| which two types of membranes can be used in southern blotting? |
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Definition
| nitrocelluose or nylon membranes |
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Term
| name a DNA sequencing method based on the principle of primer extension |
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Definition
|
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Term
| name the 5 things DNA sequencing requires |
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Definition
Single stranded DNA template
primer
dNTPs
ddNTPs (low conc)
DNA polymerase |
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Term
| name a process that amplifies DNA in an exponential chain reaction. |
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Definition
| PCR polymerase chain reaction |
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Term
| how many steps are their in PCR? |
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Definition
3.
DNA is heated to 90-95 C
A primer is annealed at temperature 50-65C
temperature increased to 70C to let thermostable DNA polymerase to synthesise 2 new strands |
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Term
| Taq is an example of a thermostable DNA Polymerase. where are these isolated from? |
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Definition
bacteria that live in very hot places like hot springs
eg. thermus aquaticus (Taq) |
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Term
| how are amplified PCR products seperated? |
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Definition
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Term
| 2 features of PCR are sensitivity and specificity. explain them a little |
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Definition
sensitivity - theoretically only one target molecule is required
specificity - both primers must match target |
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