Blog #5: DNA Extraction and Amplification

Hello!

The last few weeks of my summer research have been dedicated to the third component of my project, analysis of bacterial community composition. This is done through T-RFLP, or terminal restriction fragment length polymorphism. In order to do this assay, I first had to extract DNA, and I am still in the process of making sure I can get good amplification so that I know I have enough DNA with which to work. I am leaving campus tomorrow to prepare for a family trip, so I probably won’t be able to do T-RFLP until I resume research in the fall. However, I have still gotten some exciting results despite some setbacks in this part of my project.

DNA extraction took about a week (week #7) since I needed to extract DNA from eight nitrocellulose filters and it took about a day’s worth of lab work to perform extractions on two simultaneously. This whole process basically involves removal of DNA from filters and separation of the DNA from other organic compounds. This is done with both physical and chemical methods. First I cut up the filter into little pieces and suspend them in extraction buffer. Then lysozyme is used to lyse or burst the bacterial cells trapped on the filter. The addition of tiny disruption beads and incubation for a total of an hour loosens up the organic matter so that it is no longer attached to the filter. Proteinase K and additional heating in a hot water bath allow for digestion of proteins so that they are easier to remove from the DNA. After this, I do a series of extraction and centrifugation steps to obtain my final DNA template. I use sodium acetate, alcoholic compounds (eg phenol), and centrifugation to separate my sample into an aqueous DNA layer and a hydrophobic layer. This process is repeated until I end up with about 40 μl of DNA. A Nano-Drop analysis then allows for determination of the quantity of nucleic acids and the ratio of DNA to proteins/phenol to make sure I have a good amount of DNA (at least 500 ng/μl) and that it is pure enough to analyze.

After extraction, PCR amplification comes next. PCR stands for polymerase chain reaction and involves several cycles of heating and cooling of DNA so that it is replicated to about 230 of its original concentration, hence the term amplification. The three main steps involved are denaturation, annealing, and extension. Before all this however, a series of reagents must be added to DNA to enable the necessary reactions to occur. Since there are many reagents and one often works with several DNA template samples at once (an in replicates), it is best first to make a “Master Mix” of all the reagents by simply multiplying the amount of each reagent needed based on the number of samples used. In my case, I am looking for amplification of the 16S bacterial gene (and thus dilution of non-16S fragments), which is about 500-600 base pairs long. In order to do this I prepare 50-μl PCR samples, which each contain 49 μl of Master Mix and 1 μl of diluted DNA. The reagents needed for preparation of the Master Mix are: DEPC-H20 (or nuclease-free water), 10x or 5x buffer (depending on the Taq used), Mg+2, dNTPS, 27 forward primer, 519 reverse primer, Taq Polymerase, BSA (bovine serum albumin), and lastly, the DNA template I got through extraction.

I would like to explain the purpose of the reagents used in PCR so you can have a better idea of why they are necessary. The water is used to dilute everything else. The 10x buffer creates optimal conditions for PCR. There are many varieties of Taq Polymerase, some which contain Mg+2 and some that do not; the brand Takara ExTaq does not so I have to add Mg+2 separately in the form of MgCl2 (magnesium chloride). This is because Taq Polymerase requires Mg+2 in order to function properly. Taq polymerase is so named because it is basically DNA polymerase that has been isolated from Thermus aquaticus, a bacterium that is found in hot springs and whose enzymes can thus tolerate high temperatures. Taq polymerase interacts with dNTPS, or deoxy nucleotide triphosphates (the building blocks of DNA), by cleaving of two of their phosphates. This allows the free oxygen of the remaining phosphate to attach to the free –OH group of the primers, which thus “prime” the formation of a new strand alongside the single strand of the template after strand separation through denaturation. The bond that forms between the cleaved former dNTP and the nucleotide before it is known as a phosphodiester bond. This accounts for the sugar-phosphate backbone of the DNA ladder, and the nucleotides are its “rungs”. The last reagent, BSA, acts a stabilizing agent for the enzymatic activities involved in PCR.

I plan to wrap up my blogs with some information on gel electrophoresis and T-RFLP, as well as the general outcomes of this project. Until next time!