Sunday, 8 August 2010

Behind The Scenes....

This blog will mark the trials and tribulations that I will experience whilst carrying out my MSc Research Project.  This will be a continuing thread that I will update periodically to give you an insight of a "working progress" project, based in a molecular biology lab.  Regrettably, for now, the defining specifics of this research shall remain undisclosed.

The gist, however, is as follows:

Infection by a particular anaerobic bacteria (hereinafter called "Annie") results in different clinical presentations, ranging from a simple, easily treatable symptom right through to the opposite end of the spectrum, presenting as a fatal systemic disease.

What I want to find out is if there is a particular type of, or particular groups of, Annie that cause a certain disease presentation, that is/are not found in any other clinical presentations.  It may just be that all types of Annie are found in all types of clinical presentations of the infection, but knowing this information will help us to understand the pathogenicity of this bacteria and create possible prospects for early detection, disease maintenance and treatment.

So how am I going to do this?

Well, I have begun the early stages of this project already so I will explain with pictures below:

I'm going to be using a modified version of the technique Polymerase Chain Reaction (PCR), called Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) to look at the patterns of Annie's DNA in all the samples from the different clinical presentations, that I will be looking at.  So, first things first, I need to grow Annie so I can extract the DNA, and here's what she looks like fully grown....

Three colonies of Annie in Fastidious Anaerobe Agar
From this stage, I scoop up the colonies with a sterilised loop and swish the Annie masses into individual tubes of distilled water, one tube per subject.  It is absolutely paramount, when dealing with DNA, to use new and sterilised tools in between sampling - this avoids any cross contamination of samples which would impinge your overall results.

To begin the DNA extraction, the cells need to undergo some form of shock, to break the cells up and encourage release of the DNA.

Annies in just-boiled water.
The technique used in my molecular biology lab is to freeze shock them at -80°C and them subject them to boiling water (a freeze and heat shock method).

The beauty of working with PCR is that at any stage of the preparation process, if you find you run out of time to complete all the stages of the set-up, the samples can just be popped into the -20°C freezer and then retrieved whenever you're ready to carry on.

For some in my lab, setting up the PCR reaction using the DNA at this stage has produced some nice results, however, whether it be the modified PCR technique that I'm using, or it's just that the Annie's I'm using are a bit more robust, I have to purify Annie's DNA using the phenol-chloroform DNA extraction method to get any results.

Once the DNA is satisfactorily prepared, the next step is to create the reaction mix.  The PCR mix essentially contains all the ingredients I will need to make 1000's of copies of the DNA; nucleotides (the A, T, G & C's needed to make a copy strand), heat stable DNA polymerase enzyme Thermus aquaticus (Taq) polymerase (to bind the nucleotides to the template DNA strand), ERIC primers (markers that bind to the two template strands of Annie's DNA to indicate a starting point for replication), magnesium, buffer, water and, of course, the DNA sample (unless preparing a negative control).  A lot of the products are bought-in from a company as pre-prepared "Mastermix", so all I need to do is to add the DNA, the primers and a little sterilised distilled water.
This is a PCR machine, (right = lid open) where the tubes containing the DNA and reaction mix are placed.

It is known as a Thermal-Cycler because the silver plate heats up and cools down through cycles of varying temperatures, specific for certain stages of reaction.  

Stages of the reaction that require specific - and different - temperatures are:
  • splitting the double helix of DNA into single stranded DNA (opens sites for primers)
  • binding of the primers
  • replication of the DNA using Taq polymerase
Samples loaded into the PCR machine

The samples are loaded into the PCR machine (opposite) and the cycle is repeated again and again until 1000's of copies of DNA are made; a process that can take anywhere from around 2, or in my current case 7, hours. 

While the reaction is continuing on it's merry way, I prepare the agarose gel in which I intend to run my samples on.  This requires dissolving agarose into a 1 x tris-borate-EDTA (TBE) solution in the microwave.

Setting the gel.
Tape is carefully placed on the ends of the gel mould to create a water-tight seal (left). 

"Combs", which make the "wells" of the gel that house each individual sample, are put in place and the agarose gel solution is poured into the mould (right).  The gel usually takes around 20-25 mins to set.

Once the PCR reaction is complete and the gel has set, the tape is removed from the mould and the gel can be placed into the electrophoresis chamber (left).  
The combs are removed and the electrophoresis chamber is flooded with TBE to completely submerge the gel (right).

The number of wells in the gel (and thus teeth in the comb) need to accommodate each of the subjects sample, a DNA ladder (to which we compare product band sizes), a negative control (i.e. contains all the ingredients of the reaction mix except DNA) and a positive control (a sample known to produce a positive result that is used to prove the reaction has worked).

Agarose gel electrophoresis works on the principle that DNA is negatively charged, so when a current is applied to the uncharged agarose gel containing the samples, the DNA will "travel", by attraction, to the positively charged anode.  It is, therefore, important to load the samples at the negatively charged cathode end.  The distance that the DNA strands travel depend on their individual length, the smaller the stands, the further they travel towards the anode.

Samples with loading dye are loaded in the gel
In order to visualise the DNA products, before loading them, a "loading dye" is added to the mix.  

This is important because I need to be sure the samples are travelling in the correct direction, as well as needing to gauge how far the samples have travelled.

In the photo opposite, the samples are visible and the wells are more prominent too.

The first well (I load from left to right), will always contain the DNA ladder I mentioned earlier.  In this case I use a 1000 base pair (b.p.) ladder with markers from 100b.p. right through to 1000 b.p. and these appear in both rows of wells.

(Do ignore the basketball players below the chamber, that's just a magazine to soak up any buffer spillage).

The lid is then put on the gel electrophoresis chamber (left) with the black wire (cathode) at the same end as the loaded samples and the red wire (anode) at the opposite.

A charge is then run through the whole chamber, provided by a power pack at the back.

It is always advisable to check the samples are running in the correct direction before leaving it for the designated time.  Effective running time varies as this depends on the samples and the voltage running through it, however, a good indicator is observing that the samples have run two thirds of the way through the gel.

When the gel is ready, it is then soaked in Ethidium Bromide (below).  This binds to the DNA strands throughout the gel and allows visualisation when viewed using ultraviolet light.

Agarose gel (with samples) soaking in Ethidium Bromide

and here are the results:

Gel results viewed with ultra-violet light

This image is exactly the same layout as you see the gel in the Ethidium Bromide.  As you can see, the first lane in each row is the DNA ladder, the first few lanes thereafter in the first row are blank....yes, you guessed it, it didn't work!!  The two right-most lanes you can see with some band patterns are the positive controls, the first using Escherichia coli with universal primers, the second is Escherichia coli with the ERIC primers.

The second row with two lanes showing single bands is an unrelated test (somebody else's normal PCR run, that worked - good for them!!).

To conclude at this stage, the ERIC primers definitely work due to the positive result with Escherichia coli, however, there is something going wrong with the Annie DNA or the new method being used.

The above results were the first set of results that were tried without phenol-chloroform DNA extraction, so for the next run, I tried with this and still got the same results.

However, simultaneously, I ran another lot of samples with a tweaked PCR method.

To be continued....

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