PCR Technology
1.Introduction
Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a rapid, inexpensive and simple means of producing relatively large numbers of copies of DNA molecules from minute quantities of source DNA material--even when the source DNA is of relatively poor quality.
PCR involves preparation of the sample, the master mix and the primers, followed by detection and analysis of the reaction products. These steps are discussed below.
2.Sample Preparation
PCR is very versatile. Many types of samples can be analyzed for nucleic acids. Most PCR uses DNA as a target, rather than RNA, because of the stability of the DNA molecule and the ease with which DNA can be isolated. By following a few basic rules, problems can be avoided in the preparation of DNA for the PCR. The essential criteria for any DNA sample are that it contain at least one intact DNA strand encompassing the region to be amplified and that any impurities are sufficiently diluted so as not to inhibit the polymerization step of the PCR reaction.
Although any protocol is acceptable for PCR purposes, it is often best to use the fewest steps possible in DNA preparation in order to prevent accidental contamination with unwanted DNA. Usually a 1:5 dilution of the sample with water is sufficient to dilute out any impurities which may result from the purifying protocol.
The simplest method of isolating DNA from cells is as follows:
(1)Cells can be obtained by using a toothpick to scrape under the fingernails, swabbing the inside of the mouth or from the roots of plucked hairs. Regardless of source, cells are resuspended in 20 ul of water. Skip to step four.
(2)If you are using cells suspended in media, centrifuge at 1200- 1500Xg for 5 minutes. Resuspend the cell pellet in 1 ml of phosphate buffered saline (PBS) and repellet by spinning at 1200- 1500Xg for 5 minutes. Repeat. These PBS washes remove medium, and its inhibitory factors, from the surface of the cells. After the last wash resuspend the cell pellet in 20 ul of distilled water. Be aware that too much cell debris can inhibit the PCR reaction. If this happens, it may be necessary to further dilute the DNA sample. Go to step four.
(3)For bacterial samples take a toothpick and scrape the teeth, or swab the throat, ears or between the toes. Resuspend material in 500ul of water. Freeze and thaw sample three times with vigorous shaking or vortexing between repetitions to break the bacterial cell wall. Although not all DNA will be released from the cells, there will be a sufficient quantity for PCR. Go to step four.
(4)Place the sample in a 95oC heating block, or in boiling water, for 5 minutes. This step inactivates the DNase molecules that are found in the sample preparation. If left intact, DNase could clip the desired DNA template molecule into fragments which would be unsuitable for PCR. If there is very little DNA in the sample preparation, the DNA can be concentrated by ethanol precipitation. The sample is now ready for PCR.
DNA samples for PCR--regardless of preparation method--are generally run in duplicate in order to provide a control for the relative quality and purity of the original sample. Adding a small amount of DNA to the control just after the master mix step allows the detection of anything in the completed sample prep which would inhibit the PCR reaction.
3.Preparation of Master Mix
The Master Mix contains all of the components necessary to make new strands of DNA in the PCR process. The Master Mix reagents include:
| Final Conc. | Component | Purpose |
| / | Water | / |
| 1X | Buffer | keeps the master mix at the proper pH so the PCR reaction will take place. |
| 200uM | Deoxynucleotides | provide both the energy and nucleosides for the synthesis of DNA. It is important to add equal amounts of each nucleotide (dATP, dTTP, dCTP,dGTP) to the master mix to prevent mismatches of bases |
| 0.2-1.0uM | Primers | Short pieces of DNA (20-30 bases) that bind to the DNA template allowing Taq DNA polymerase enzyme to initiate incorporation of the deoxynucleotides.Both specific and universal primers can be used. |
| 2.5U/100ul | AmpliTaq polymerase | A heat stable enzyme that adds the deoxynucleotides to the DNA template |
| 0.05-1.0ug | Template DNA | The DNA which will be amplified by the PCR reaction. |
4.Notes on the Master Mix
The Master mix buffer is often stored as a 10X stock solution (100 mM Tris-HCL, pH 8.3, 500 mM KCL, 1.5 mM MgCl2) which is diluted to 1X for use. Both the Master mix buffer and the purified water can be stored at room temperature. Store deoxynucleotides, primers and Taq DNA polymerase enzyme at -20℃.
Although 100ul of master mix per reaction is generally used, it is possible to use as little as 25 or 50ul to save on cost of reagents. Regardless of the total volume, be certain to keep the final concentrations of reagents constant.
Master mix reagents can be optained from a variety of companies. Often the initial concentration of the reagent will differ depending on which company produced it. It is easy to figure out how much stock reagent to use by following a simple formula:
(initial concentration) X ( volume needed ) =(final concentration) X (volume of sample)
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