BIOL114 - Lecture Techniques to study gene/ DNA structure

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  • Created by: Katherine
  • Created on: 15-03-16 13:09
What are agarose gels typically referred to as?
Submarine gels
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How is gel elctrophoresis conducted?
You have a tank in which you pour agarose gel, and you have a well former at the top which creates these wells.
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What is gel electrophoresis used for?
To separate lots of different fragements, and they separate based on size
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What else is in the gel electrophoresis?
A dye, and usually a sugar such as glycerol.
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Why is glycerol usually added to the solution?
To make it more dense
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The gel is covered by what?
A buffer
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Why do we load DNA on the end that is nearest to the cathode?
Because DNA is negatively charged - this is due to the phosphate group
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The agarose creates a mesh which does what?
Size fractionates the DNA - so very big bits find it hard to move through the gel.
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What are markers?
These are standard DNA molecules which are at the side of the gel as we know the exact size of these. We use these to compare the sizes of unknown fragment sizes.
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You'll measure the sitance and plot it against what?
The known value
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When you load the DNA onto the gel, we use a dye, why?
This is so it can be easily identified and so you can visualise where the bands are.
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What used to be used as a dye, and why is it not any more?
Ethidium bromide, it is a carcinogen.
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What is now used to dye the DNA fragments?
Gelred - both compounds intercollate in the DNA and under a UV light source, they fluoress
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What does a restriction enzyme do? How does it work?
It recognises particular bases, and will cut double stranded DNA
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Why might you have a theorhetical number of fragments that is bigger than the actual number?
Because they co-align in the gel (co-segregate)
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Why do we use a restriction map?
It order to see how closely organisms are related to each other, or to see if someone is carrying a mutant gene.
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What is RFLP
it shows that the restriction enzyme will cut at a particular position
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Why is restriction enzyme dgested huamn DNA not always useful?
It often appears as just a smear because there are several hundered thousand bands.
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What is PCR?
A method which is used to amplify DNA.
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How many steps does PCR consist of?
3 steps
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What are the 3 steps of PCR?
DNA is heated to 90-95'C. You put in a primer which attaches and is annealed at about 50-65'C. The temp is raised to 70'c to allow thermostable DNA polymerase to synthesise 2 new strands.
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Why is the DNA initially heated?
To break the hydrogen bonds so you're left with two separate strands of DNA
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What are primers?
Short pieces of synthetic DNA
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Why is a primer required?
Becuase more DNA will nly be made if it starts with double stranded DNA. iT NEEDS THAT VERY SHORT PECE OF DOUBLE dna IF IT'S GOING TO SYNTHESIS MORE DNA.
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What determines the temperature that the reaction is going to take place?
The number of A,G,C and Ts there are because you have 3 hydrogen bonds between a G and a C pairing, and two between A and T.
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Do you need a higher or lower temperature to get it to stick together if it is G-C rich?
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What is the enzyme used to allow DNA to polymerise?
Taq - this works at a very high temperature that enables this process to take place
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Why does Taq not denature?
It is from bacteria that live in hot springs. (Thermus aquaticus) It is found in organisms that live in incredibly high temperatures.
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How do you make DNA using PCR?
You start with your genomic DNA and add your Taq, primers and dNTPs.
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What re the dNTPS?
The building blocks that put the phosphate backbone back together.
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What is the first step of PCR?
Heat it up to a high temperature. Pull the two DNA strands apart.
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What is the second step of PCR?
You put in a primer at the 3' end. You can only make DNA in the 5'-3' direction. So you need a primer that sits at the 3' prime end of the strand that you're trying to make the complementary strand to.
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What is the third step of PCR?
Increase the temperature to 70' where the DNA polymerase works at an optimum temperature.
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After 2 cycles of PCR, how many molecules do you end up with?
4 molecules.
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What are the features of PCR?
It is very sensitive, so only one target molecule is required - useful in forensics when you have a very small amount of DNA. Specificity - we can provided this as the primers we design must match the target
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Why can the sensitivity of the PCR be bad?
It is easy to contaminate it.
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Why do we use PCR?
To amplify somethning. if there's a particular sequence which we want to clone. Fo species identification (e.g. the meat scandal). For identification of disease alleles. In forensics and in gene expression.
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What is Southern blotting?
It allows us to look for particular DNA sequences in a very complex mixture
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What is the process of southern blotting?
It combines some of the methods we've already looked at - it's taking DNA and doing a restriction digest and gel elctrophoresis.
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What is the exact blotting tage of SOuthern blotting?
You create a block and create a wick where solution moves through the agarose solution, through the sponge and up to the nitrocellulose membrane block.
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What is the purpose of Southern blotting?
It is a way of rensfering DNA in the exact pattern you've seen on the gel electrohporesis.
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What is the second step of Southern blotting?
Now that you have a copy of the DNA on the membrane, if you know the sequence of the gene your looking for, you can radioactively label it. You put the radioactively labelled gene in a bag with the blot and it will bind to the bits that you were loo
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It's about using different probes to highlight the thins that are in there
You then put the nitrocellulose mix next to an x ray film and put it in a heavily fortified case , where ever you have radioactive links, you will get a shadow on the gram .
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What does dideoxynucleotide sequencing require?
Single stranded DNA template, primer, dNTPs, ddNTPS, DNA polymerase.
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What is dedeoxygnucleotide sequencing based on?
The principle of primer extension
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What happens in DNA syntheiss?
You make a complementary strand to the DNA your syntheisis
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The process will only continue if you have two things, what are these things?
A hydroxyl residue and if you have on the nucleotide coming in, phosphates available at carbon 5. If you lose this you can't extend as it is required in order to form the backbone.
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What does it mean if the OH residue is not available?
YOU cannot form the phosphodiester bond and DNA synthesis will stop.
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If you're tryig to ind out the sequence of a piece of DNA, what do you do?
You make a copy of it where it stops after each of the bases. Each of the didexoynucleotides are fluorescently tagged, and has an H residue, rather than an OH.
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What happens everytime the dideoxynucleotide jois?
Synthesis will stop because there is not an OH.
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Then, with the set of DNA, what can you do?
You know that the last base pair you put in in flurescently tagged. So you can run all the fragments down a gel (it's not an agarose gel). You sine a lazer through it and you can see what the first nucleotide is in a complementary base pair.
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What is the most important thing to rembmer about this?
That this is the complementary strand, so the actual sequence is the opposite of this.
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Can you combine DNA synthesis with PCR?
Yes, you carry out DNA synthesis in a PCR setting.
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How can this be done?
We make sure that the DNA polymerase can withstand high temperatures.
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In order to do DNA synthesis, how many primers do you need? And why?
1 because you only need one strand.
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Card 2


How is gel elctrophoresis conducted?


You have a tank in which you pour agarose gel, and you have a well former at the top which creates these wells.

Card 3


What is gel electrophoresis used for?


Preview of the front of card 3

Card 4


What else is in the gel electrophoresis?


Preview of the front of card 4

Card 5


Why is glycerol usually added to the solution?


Preview of the front of card 5
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