Introduction to PCR
Virtually all DNA/RNA based tests require three basic steps.
In this first step, all nucleic acid material (DNA/RNA) must be removed and isolated from the specimen to be tested which may be hair, blood, tissue, or other material.
All biological samples contain lots of nucleic acid material. Only a small portion of all of this nucleic acid material consists of the "target"; the nucleic acid sequence being tested for. Without this step, the amount of non-target material present in the sample would make the target material of interest virtually impossible to detect. The process of amplification increases the number of target sequences to a level that is detectable. PCR, or Polymerase Chain Reaction, is the most widely used method for amplifying the nucleic acid target. Thermal Gradient's technology is centered around performing this step faster, less expensively, and in a highly automated fashion.
Once there is enough target in relation to the rest of the nucleic acid material, the final step of detecting the absence or presence of the target can be performed.
How PCR Works
What is the problem?
Pretty much all "nucleic acid" tests (DNA, RNA) are "needle-in-a-haystack" problems. The biologic specimen to be tested (hair, blood, sweat, etc) contains all sorts of nucleic acid material (the "hay") and very little of the target material you are looking for (the "needle") such as that for an infectious disease. All the hay makes it difficult to see the very few needles that may be present. The typical approach to solving this sort of problem is to find a way to reduce the amount of hay so that there is less of it to hide the needles that are of interest. Polymerase Chain Reaction (PCR) takes the opposite approach. It causes the target material to multiply until there is enough of it to be distinguishable from the hay.
PCR was developed in 1983 by Kary Mullis. It is now the most commonly used amplification technique in nucleic acid testing in a wide field of applications such as diagnostic testing, forensics, genomic research, and genetic disorders and hereditary disease testing. Dr. Mullis was awarded the Nobel Prize in Chemistry in 1993 for the invention of PCR.
The PCR process starts with a buffer solution with a mix of reagents consisting of:
- Primers: Short manufactured sequences of DNA that are complimentary (uniquely matched) to the target DNA
- Polymerase: An enzyme that assists in the reconstruction of DNA
- dNTPs: The individual single components (C, G, T, A) from which DNA strands can be assembled.
The PCR sample is created by combining the reagent mix with the nucleic acid material that has been extracted from the original specimen such as blood or hair.
How To Cook
The process of causing the target material to multiply in numbers (or "amplify") is done by exposing the sample to a series of temperature changes. This is referred to as "thermal cycling." Generally, there are three temperatures in each thermal cycle; 95°C (203 °F), 55°C (131 °F), and 72°C (162 °F).
Denaturing - "Bring to a Boil"
Well not quite a boil but pretty close to it. Elevating the temperature of the sample to 95°C (203 °F) will cause the double helix structure of all the DNA strands to unwind thus creating twice as many single strands. This is referred to as "denaturing."
Annealing - "Allow to Cool"
When the sample is cooled to around 55°C (131 °F), the single strands of DNA want to recombine back into full double strands. The non-target DNA does exactly that. These single strands of DNA recombine back into their original double stranded form. However, for the target DNA, the primers are present. Because these target-specific sequences are much shorter than the original longer target stands, they attach to the target strands before the original complementary strands can reconnect.
The sample solution now contains reconstructed non-specific DNA (the "hay") in the same numbers as before. The sample also now has strands of the target DNA that are double stranded only where the primers are attached and single stranded for the rest of the sequence. Since it takes only one single strand to make this structure, there are twice as many of these single strands of the target DNA with primers as there were of the double strands of the original target.
Extension - "Simmer and Stir Gently"
Here is where the polymerase and the dNPTs comes in. When the PCR sample is brought to an intermediate temperature of 72°C (162 °F), the polymerase attaches itself to the section of the target where the double stranded portion with the primer ends and the rest of the single stranded section begins. The polymerase attracts the appropriate protein (C, G, T or A) to that location and reconstructs that base pair of the target sequence. The remaining bases of the target sequence are reconstructed in the same way, with the polymerase assembling (extending) the unfinished sequence until a fully reconstructed double stranded target is the result. Since only one single strand is required for each reconstructed new double strand and each original double strand contributes two single strands to the process, each thermal cycle results in TWICE as much target material as was present at the start of each cycle.
Since the amount of target DNA doubles with each thermal cycle, the ratio of "needles" to "hay" also doubles with each cycle. 20-40 thermal cycle PCR reactions are not uncommon. That would mean that there would be 1 million to 1 trillion times as much DNA as there was to start with.