PCR amplification of ADE1 and ADE2 from Red Yeast P olymerase C hain R eaction (PCR) is an in vitro (in test tube) method to replicate DNA. It bears some basic resemblance to the way DNA is replicated inside the cell but also differs in some fundamental ways. The power of PCR technology lies in the ability to start with tiny quantities of DNA material that are essentially undetectable and selectively amplify a region of interest into such amounts that the amplified DNA is simple to detect. The PCR process involves three steps in repeated cycles of varying temperatures. The cycling allows replication of the DNA by 1) denaturation of double stranded DNA template (but not the thermostable polymerase) at 95°C, 2) annealing of the short complementary DNA oligonucleotides (primers) to the single- stranded template at 55°C, then 3) extension of the DNA strand from the primers by the polymerase at 72°C. The thin-walled PCR tubes and the rapid temperature change of the thermocycler allow these cycles to occur very rapidly. One copy of the double- stranded template can be amplified to more than a billion (2 30 >10 9 ) with 30 cycles in 2-3 hours in a 25 µl reaction (Fig 1 from Brooker). Genetics Fall 2013 Figure 1: The 2 primers anneal to specific sites within the template DNA and 1
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PCR amplification of ADE1 and ADE2 from Red Yeast
Polymerase Chain Reaction (PCR) is an in vitro (in test tube) method to replicate DNA. It bears some basic resemblance to the way DNA is replicated inside the cell but also differs in some fundamental ways. The power of PCR technology lies in the ability to start with tiny quantities of DNA material that are essentially undetectable and selectively amplify a region of interest into such amounts that the amplified DNA is simple to detect.
The PCR process involves three steps in repeated cycles of varying temperatures. The cycling allows replication of the DNA by 1) denaturation of double stranded DNA template (but not the thermostable polymerase) at 95°C, 2) annealing of the short complementary DNA oligonucleotides (primers) to the single-stranded template at 55°C, then 3) extension of the DNA strand from the primers by the polymerase at 72°C. The thin-walled PCR tubes and the rapid temperature change of the thermocycler allow these cycles to occur very rapidly. One copy of the double-stranded template can be amplified to more than a billion (230 >109) with 30 cycles in 2-3 hours in a 25 µl reaction (Fig 1 from Brooker).
Genetics Fall 2013
Figure 1: The 2 primersanneal to specific sites withinthe template DNA and amplifya defined region of DNA that islocated between the 2 primers.
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The strains of yeast with red color have been associated with a defect in the ability to make adenine. Adenine is chemically related to several molecules that have a red color, although adenine itself does not. Normally these molecules that have a red color are converted to adenine by two different enzymes. Two genes, ADE1 and ADE2, encode these enzymes. A defect in either one of these enzymes can lead to the accumulation of one of these molecules that have red color, thus the red color yeast. In this lab you will amplify DNA from the ADE1 gene and the ADE2 gene.
The following components are required to perform a PCR:1. Source of DNA to be amplified: This starting material may be purified DNA or can be small amounts of tissue or cells. In forensic medicine, one is often forced to use small bits of tissue left at the crime scene. You will use the DNA that you isolated from a single colony of red yeast.2. A pair of short, single stranded DNA molecules called oligonucleotides that will be used to prime the replication reaction. Because of their function these oligonucleotides are also called primers. In this particular case, you will be given primers of the appropriate base sequence to amplify the ADE1 gene and the ADE2 gene.3. Thermostable DNA Polymerase enzyme: DNA polymerases are enzymes that have the ability to incorporate deoxyribonucleotides into a growing chain of DNA. For PCR, it is essential that the enzyme be heat stable (thermostable). Heat stable enzymes are generally found in
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bacterial sources that grow in very harsh environments. The particular enzyme used most often is called Taq polymerase. Thermus aquaticus grows in and around hot geysers.4. deoxynucleotide triphosphates (dNTP’s): These are the building blocks of DNA and are the molecules that the polymerase enzyme incorporates into the growing DNA chain.5. Buffer: This will make sure that the pH and ionic requirements for the DNA polymerase are met.
You will be using Ready-To-Go PCR beads to setup your PCR reactions. This bead has the dNTP’s, the DNA polymerase, and the buffer. Traditionally there would be three separate solutions to pipet (dNTPs, DNA polymerase and the buffer).
Procedure
1. Obtain from your instructor a. 2 tubes, each containing a Ready-To-Go PCR bead b. Ade1 primer mix (contains the forward and reverse primer to amplify ADE1)c. Ade2 primer mix (contains the forward and reverse primer to amplify ADE2)
2. Label 1st tube as Ade1 and the 2nd tube as Ade2. Add your group # to both tubes so that you can identify your tubes.
3. To the Ade1 tube add the following in the order listed below. Make sure to use a clean tip for each reagent.
Ade1 primer mix 12.5 l Water 7.5 l Yeast DNA 5.0 l
25.0 l (Total volume)
4. To the Ade2 tube add the following in the order listed below. Make sure to use a clean tip for each reagent.
Ade2 primer mix 12.5 l
Water 7.5 l Yeast DNA 5.0 l
25.0 l (Total volume)
5. Close the tubes and gently flick the tubes with your finger until the bead is completely dissolved.
6. Place the tubes in a microcentrifuge and spin for 1 min at 3000 rpm. 7. Place the tubes in the thermocycler. It will be started when all the groups have placed
