PCR Technology

互联网2013-09-06

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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.

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:

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.
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.
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.
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.

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      Component Purpose 
Conc.
           Water

1X         Buffer       keeps the master mix at the proper pH so the PCR
                        reaction will take place. 

200uM      Deoxynu- provide both the energy and nucleosides for the 
           cleotides    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  A heat stable enzyme that adds the
           polymerase deoxynucleotides to the DNA template.
 

0.05-1.0ug Template  The DNA which will be amplified by the PCR
           DNA  reaction.

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 -20oC.

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)

For example: I have 10X buffer, 10 mM of each nucleotide, 0.5 mM primers and Taq DNA polymerase at 5 Units/ul. I want to make one 50 ul reaction. Calculations are as follows:

10 X buffer: (10X) X (5 ul) = (1X) X (50 ul) Nucleotides: (10,000 uM) X (1 ul) = (200 uM) X (50 ul) (10mM=10,000uM) primers (500uM) X (O.1ul)= (1.0uM) X (50 ul) Since it is impossible to pipet 0.1ul accurately, a dilution needs to be made first. Add 10 ul of stock primer solution to 990 ul of water to get 5uM concentration of primers. This new primer dilution can be stored at 4oC. Calculation for 5uM stock: (5uM) X (10 ul) = (1.0 uM ) X (50 ul) Taq DNA polymerase (5Units/ul) X ( 0.25 ul) = (.025 Units/ul) X (50 ul) 2.5 Units/100ul= Since it is impossible to pipet 0.25ul accurately, a .025 Units/ul dilution needs to be made first. Add 1.25 ul stock to 3.75 ul water to get a 1.25 Units/ul concentration. Discard and make fresh with each use. Calculation for 1.25 Units/ul stock: (1.25 Units/ul) X (1 ul) = (.025 Units/ul) X (50 ul)

To make the master mix for one reaction add:

5 ul 10X buffer
4ul Each nucleotide (1ul each of dATP, dCTP, dGTP, dTTP))
20 ul Each primer (10ul of each)
1 ul Taq DNA polymerase (Total volume = 30ul)
add 15 ul of water
5 ul of template (Total volume = 50 ul)

If want to make 3 reactions, 3 X 50ul = 150ul. Use this number in the formula for "volume of sample."

Primers

A primer is a short segment of nucleotides which is complementary to a section of the DNA which is to be amplified in the PCR reaction. Primers are annealed to the denatured DNA template to provide an initiation site for the elongation of the new DNA molecule. Primers can either be specific to a particular DNA nucleotide sequence or they can be "universal." Universal primers are complementary to nucleotide sequences which are very common in a particular set of DNA molecules. Thus, they are able to bind to a wide variety of DNA templates.

Bacterial ribosomal DNA genes contain nucleotide sequences that are common to all bacteria. Thus, bacterial universal primers can be made by creating primers which are complementary to these sequences.
Examples of bacteria universal primer sequences are:
Forward 5' GAT CCT GGC TCA GGA TGA AC 3' (20 mer)
Reverse 5' GGA CTA CCA GGG TAT CTA ATC 3' (21 mer)

Animal cell lines contain a particular sequence known as the "alu gene". There are approximately 900,000 copies of the alu gene distributed throughout the human genome, and multiple copies distributed through the genome of other animal cells, as well. Thus, the alu gene provides the sequence for a universal primer for animal cell lines. The alu primer is especially useful in that it binds in both forward and reverse directions.
The alu universal primer seqeunce is as follows:
5' GTG GAT CAC CTG AGG TCA GGA GTT TC 3' (26mer)

When using universal primers the annealing temperature on the thermal cycler is lowered to 40-55 degrees C.

Sometimes primer units are listed in optical density reading (OD). If this is a problem you will need to convert to molarity using the following equations: Change optical density reading of primer to molarity (uM units)-

N = # of primer bases
SIGMA 260 =~ 10,000 X N/ m X cm
Molecular weight =~ 330 X N
OD₂₆₀ / SIGMA 260 X 10⁶ = Concentration (uM)

For example- primer is 20 bases long/ OD₂₆₀ = 10.

N = 20
SIGMA 260 =~ 10,000 X 20/m X cm = 20,000/m X cm
molecular weight =~ 330 X 20 = 6,600
10 OD₂₆₀ /20,000 m^-1 cm^-1 X 10⁶ = 50uM

Detection and analysis of the reaction product

The PCR product should be a fragment or fragments of DNA of defined length. The simplest way to check for the presence of these fragments is to load a sample taken from the reaction product, along with appropriate molecular-weight markers, onto an agarose gel which contains 0.8-4.0% ethidium bromide. DNA bands on the gel can then be visualized under ultraviolet trans-illumination. By comparing product bands with bands from the known molecular-weight markers, you should be able to identify any product fragments which are of the appropriate molecular weight.