3rd year practicals - exam paper - Question 4!!

Hi you all 3rd years !!

Today we continue to with the 3rd year practical sample exam paper:)

Question 4 Part a) Main reagents of the PCR reaction are:

DNA tamplate - I think I do not have to explain that:) DNA template contains the piece of the DNA that we wish to amplifiy.

Primers - short pieces of DNA (oligonucleotides, usually around 20-40 nucleotides) that specify which sequence of DNA will be amplifiied.

Buffer - usually provided as 10x. Diluted buffer provides optimum conditions (pH, salt concentration, coffactors etc) for the PCR reaction.

dNTP's - deoxynucleotidetriphosphate's (dATP, dTTP, dCTP, dGTP) are substrates in PCR reaction and are necessary to elongate the primers what result in PCR product.

MgCl2 - magensium chloride is essential reagent in the PCR reaction (sometimes it is icluded in the buffer solution but not always). MgCl2 (actually the magnesium divalent cations) stabilize the negtive charge of the dNTP's (remember that phosphate groups on nucleotides are negatively charged). This stabilization helps in alignment of the nucleotides during their incorporation to newly synthetized DNA strand. You can imagine it like that:)

This interaction is way more complicated (first it is not flat like that:). This is just to give you an idea what is happening:)

Enzyme - those are always DNA polymerases. There are different types of polymerases used for PCR purposes but all of them are thermostabile. Some of them are more error prone than others. Examples of the polymerase used: SigmaTaq, Takara LA Taq, KOD Polymerase and more.

Water - used as an essential solvent.

In the second part of the question you are asked to explain differentce between PCR and RT-PCR. RT-PCR can stand for either Real-Time PCR or Reverse-Transcriptase PCR. I will give you short definitions of both so you will know the main differences between them.
Real Time PCR is a very sensitive version of the regular PCR where real time analysis of the obtained product is possible. For that purpose fluorochromes are used to labell the arisen products what gives us possibility to quantify the amount of the product in whatever time during the reaction. Simply Real Time PCR is a quantitative version of the regular PCR.
Reverse Transcriptase PCR is a reaction in which cDNA (protein coding DNA sequence) is produced using the RNA. What it that means is that RNA is first used as a template to produce DNA (Reverse Transcriptase is an enzyme that uses RNA as a template to produce DNA) and then this DNA is used to amplify our sequence of interest.

Part b) Agarose is a polysaccharide obtained from agar. Agarose has wide range of applications and one of them is gel electrophoresis. Agarose can form gel like structures with the pores depending on the agarose concentration (usually expressed as %). Agarose electrphoresis is mainly used for the separation of the nucleic acids like DNA or RNA (but it can be used for protein separation as well). Simply nucleic acids loaded onto gel will migrate through the gel pores depending on their size. Small nucleic acids molecules will migrate faster (they go easier through the pores) than the big ones.
Ethidium Bromide (usually abbreviated as EtBr, do not confuse with ethylene bromide) is an intercalating agent. Interacts with the DNA is mediated through hydrophobic interactions between aromatic rings of EtBr and nucleotides (nitrogonous bases of nucleotides contain aromatic rings). See figure below:

Taken from http://www.madsci.org/posts/archives/1999-02/919869466.Mb.1.jpg

Ethidium Bromide is a fluorophore which have maximum adsorbtion around 300nm (it can be visualised with UV lamp and has an orange like colour). Its adsorbtion is even greater when bound to DNA. Remember that EtBr is a mutagen because its intercalation cause change in DNA structure and may interfere with DNA metabolism processes like DNA replication

Part c) In this question you are asked to estimate size of the unknown DNA molecules using the standard curve. First you have to calculate the -logRf (you can calculate the logRf as well that do not change enything) and put them together in the Table:

Then we plot the -logRf vs Molecular Size (bp). Using the standard curve you read out the molecular size of the unknown samples using the -logRf. I have used equation to calculate the sizes of the unknown samples but you have to do that manually (just remember to use as accurate scale as possible). Notice that unknown sample C gives me a negative value (which does not make any sense because DNA molecule can not have negative mass:). The reason for is that Rf of this sample is outside the range of standard curve:) Be careful and alert, it often happens that there is something tricky about the questions, lectures want to confuse you, so you have to be sure and confident about your calculations and answers:) Do not let them win:) In this case you have to indicate that Rf of this sample is outside the range and it is not possible to estimate its molecluar mass.

That is all for today.

Remember that if you have any more problems or if something is not clear or understandable, just let me know. Leave a comment or send me email to m.kliszczak1@nuigalway.ie.

Tomorrow will take care of the Question 5.

Cya

Maciek