POLYMERASE CHAIN REACTION AND AGAROSE GEL ELECTROPHORESIS
INTRODUCTION: In working with DNA, sample size becomes a problem. A researcher needs
sample DNA to work with for identification or experimentation. Until the advent of PCR, the only way to purify a DNA sequence was through biological amplification in cultured cells. The entire process encompassed a period of several days and required ligating the desired sequence into a vector, transforming the recombinant vector into an appropriate host cell line, culturing the transformed cells, purifying the construct from the transformants, and releasing the fragment from the vector molecule. In contrast, PCR uses enzymatic amplification to increase the copy number of any DNA fragment of up to approximately 6000 bp. Using data from a database of sequence-tagged sites (STS’s), a researcher anywhere in the world can use PCR to assay for a particular STS in as little as 24 hours. PCR is based on the phenomenon of primer extension by DNA polymerases, which wasDiscovered in the 1960’s. First, oligonucleotide (15-20 bp) primers are synthesized that are complementary to the 5’ end of each strand of the sequence-tagged site. The two primers are mixed in excess with a DNA sample from which the target sequence (STS) will be amplified, along with a heat-stable Taq DNA polymerase from Thermophilus aquaticus (a bacterium which inhabits hot springs). The four deoxyribonucleoside triphosphates are also provided, one or more of which may be radioactively labeled or labeled in another, visual way. The reaction mixer is than taken through multiple synthesis cycles (which will be described in the lab protocol). Twenty-five rounds of the synthesis produce 1,000,000 new copies of the STS in less than 3 hours, for example. In this lab, we will be amplifying a sequence of DNA obtained from cyanobacteria and onion from the previous lab. In the process of amplifying the cyanobacterial and onion DNA, we will also hopefully insert selected genes, hsp16.6 and/or cox II into a percentage of the amplified DNA. The hsp16.6 gene is a heat-shock gene, and the coxII is a gene intron, which seems to enable plants usually grown in warm climates only to survive colder temperatures in the northern climates. The product of our PCR will then be electrophoresed to analyze results. Agarose gel electrophoresis is used to separate DNA fragments of different sizes. Since we know that the selected insert genes’ sizes are ~450 bp for hsp16.6 and ~650 bp for coxII, we may be able to determine if the genes have actually been taken up and inserted into the sample genomes. Seeing them separate out in the gel at the appropriate distances from their origin of separation will verify this and will be determined in the successive labs.
MATERIALS AND METHOD: Follow directions on the following protocol pages and note any modifications.
PCR Amplification of DNA
1. Thaw all components (except Taq Polymerase) on ice 30 min before the lab.
2. Combine the following components for each reaction (on ice) in an Eppendorf tube:
|
Tube (1.5 ml Eppendorf) |
Distilled water |
10X PCR buffer |
dNTP |
5 unit/µl Taq Polymerase |
Total |
|
Wizard Mix |
284 µl |
36 µl |
36 µl |
4 µl (ask TA to pipet enzyme) |
360 µl |
3. Vortex and centrifuge for 30 seconds at 13,000rpm.
4. Combine the following for each reaction in a PCR tube(50 ul total reaction volume).
|
Tube |
Wizard Mix |
DDH20 |
A plasmid containing hsp16.6 gene |
Cyanobacterium genomic DNA |
A plasmid containing cox II gene |
Onion genomic DNA |
Primers (F and R) for hsp16.6 gene |
Primers (F and R) for Cox II gene |
|
1 (Negative control) |
47 µl |
1 µl |
1 µl primer Fh 1 µl primer Rh |
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|
2 hsp16.6 gene control |
47 µl |
1 µl |
1 µl primer Fh 1 µl primer Rh |
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|
3 hsp16.6 gene |
47 µl |
1 µl |
1 µl primer Fh 1 µl primer Rh |
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|
4 (Negative control) |
47 µl |
1 µl |
1 µl primer Fc 1 µl primer Rc |
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|
5 cox gene control |
47 µl |
1 µl |
1 µl primer Fc 1 µl primer Rc |
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|
6 cox gene |
47 µl |
1 µl |
1
µl
primer Fc |
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|
7 Positive control |
47 µl |
PUC18 plasmid provided in the kit 1 ml |
PUC18 primers provided in the kit 1 ul F +1 ul R = 2 µl total |
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. Run the following program (about 2.5 h):
Initial Step
 | 94 oC 3 min - |
Next 30 cycles:
| 94 oC 1 min
| 58 oC 1 min (or annealing temperature appropriate for
particular primer pair)
| 72 oC 1 min (if product is <500 bp), 3 min (if product is
>500 bp) for 30 cycles. | |
Program a final extension at
| 72 oC for 7 min. Hold at +4°C. |
5. Take 10 µl of PCR product from each tube for gel electrophoresis (Refer to the protocol for agarose gel electrophoresis).
Materials:
sterile water
10X amplification buffer with 15mM MgCl2
10 mM dNTP
50 µM oligonucleotide primer F (forward)
50 µM oligonucleotide primer R (reverse)
5 unit/µl Taq Polymerase
template DNA (1 µg genomic DNA, 0.1-1 ng plasmid DNA) in ddH20
These web pages were prepared for Botany 415/515
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Kenneth G. Wilson
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Susan R. Barnum
|
Feng Fang
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RESULTS: After PCR was completed, the contents of the tubes were loaded into an agarose gel and
electrophoresed. In lane numbers one and four, there was no evidence of any segment. This was
expected, because there was no gene contained in either preparation, as these tubes served as
negative controls. If there had been any banding in either of these tubes, our results would have
been invalid. In lane 2, there was distinct banding, which was also expected, because this con-
tained the known gene control, hsp16.6. Lane number three contained the questionable cyano-
bacterial isolated gene hsp16.6, and results showed banding very similar to the gene control for
the very same gene. In lane 5, a very light band showed up, but the position of this band was
inconsistent with the expected results of a known size of ~650 bp, as this was the positive control
for the cox II gene. Lane number six, which should have had an isolated band of DNA from the
onion cell was empty. The onion cell DNA coxII gene had not been successfully isolated. Lane
number 7, the positive control showed banding consistent with the expected PUC18 plasmid
provided by the kit manufacturers, whose appearance in the lane proved that the system was
working (that the electrophoresis was actually occurring as expected). The gel was photographed,
and the resulting picture is shown below.
Use Web address for gel picture.
DISCUSSION: The results obtained and shown above are consistent with what was expected, except
for the failure of the onion DNA isolation. There may have been too much RNA left attached to
the DNA, which did not allow the primer to anneal to the coxII gene so that it could have been
isolated from the onion DNA. Group 2 researchers obtained the results expected as shown on
the gel photo, and the other groups did not. There may have been a variation in the way
their lab techniques were performed.
If this lab can be performed in a high school classroom, the following thought questions may be used:
1. Why are both forward and reverse primers required to amplify DNA?
2. What is the purpose of the positive control?
3. Why use a negative control?
4. What is the purpose of the 94-degree step in the PCR cycle?
5. Why are nucleic acids negatively charged?
6. Why do large DNA fragments run more slowly than small DNA fragments through a gel?
7. How does ethidium bromide make DNA visible on gels?
CONCLUSION: The lab procedures performed here illustrate the value of both polymerase chain
reaction and agarose gel electrophoresis. The experimental gene was isolated, amplified, and its
presence noted by the band in the gel. This is in no way, however, positive proof that what the ex- perimenters saw was, in fact, the gene in question. In the succeeding experiments, additional pro-
cedures will help validate or nullify the conclusions arrived at in this lab.
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