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NOTE: Reading all of the context is not completely necessary - it would be beneficial for the reader in terms of their own base knowledge and in understanding the workings of the project.








DNA structure



In order to understand how PCR or RT-PCR works, the DNA structure must first be noted.

The DNA molecule is the entire strand that we normally associate DNA with. The units that make up the DNA are called nucleotides – nucleotides have a sugar component, a phosphate group, and a nitrogen group.

The sugar and the phosphate always remain the same. However, the nitrogen group can vary between four different types of nucleotides: adenine (A), guanine (G) [these two are the purine groups], cytosine (C), and thymine (T) [which are the pyrimidines].

The nucleotides are bonded together to form a long chain or strand. Two of these strands are hydrogen-bonded together to form a DNA molecule.

Each rung of the DNA “ladder” is shared by two nucleotides; one purine and one pyrimidine – this is the only way to obtain equal length rungs. Furthermore, the purines and pyrimidines are specific in terms of their partners on the ladder. If the purine is guanine, them the corresponding pyrimidine must be cytosine. If the purine is adenine, the pyrimidine must be thymine. Due to this specificity, the DNA strands are complementary to each other.


The concepts of complementary pairing and DNA strand formation is used in PCR.

EXAMPLE: The following two strands are complementary

G A T T C G T A C G Strand 1
C T A A G C A T G C Strand 2


The DNA strands also have two leading ends – a 5’ end and a 3’ end. The ends are either the “leading” end (5’) or the “tail” end (the 3’ end). The ends are recognized by the labeled carbon of the sugar group of the nucleotide.




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PCR



Although I mentioned reverse transcription PCR and Real-time PCR earlier, it is necessary to first understand how BASIC PCR works in order to understand real-time PCR, which is an extension to the basic PCR (this is analogous to understanding how to add and subtract before one can understand algebra).

PCR, a technique developed about 20 years ago, uses a series of temperature cycles to rapidly produce vast quantities of a specific segment of DNA from only minute initial amounts. Example: a scientist may be dealing with a whole segment of a DNA strand, but may only want to reproduce a specific region that contains a specific gene that he/she is studying.

MATERIALS
The reaction requires the following basic components, which are mixed together into a single reaction tube:
1. Template DNA (a sample of the DNA that you wish to replicate)
2. dNTPs (Deoxyribonucleic Triphosphates – are the units of which the DNA molecules are constructed of, each carry a differing nitrogen base. The four dNTPs are: dATP, dCTP, dGTP, dTTP)
3. Two Primers – a forward and reverse primer (Primers are strands of nucleic acid that are starting/ending points for DNA replication; they define the region of DNA to be replicated in PCR).
4. DNA polymerase (enzyme that replicates DNA; there are many different kinds but the most commonly used is the Taq DNA polymerase because of its thermo-stability)
5. Buffer (helps to stabilize the pH and may be the alternative to magnesium chloride)
6. Magnesium (activates the DNA polymerase)
7. Other optional additives (such as inhibitors)
8. Thermal Cycler (a programmable device that cycles between specific temperatures for set periods of time – used for PCR).Primers are specially designed to anneal to spots on the DNA template that encompass the target region.


PROCESS
The PCR process occurs through a series of cycling of different temperatures that are
held for certain lengths of time. The cycling conditions/parameters differ from reaction to reaction, but the basic cycling parameters are set for three basic steps: 1) denaturing the template DNA (separating the double stranded DNA), 2) annealing the primers (attaching the primers to the DNA template), and 3) extension of DNA (the replication of DNA).



POLYMERASE CHAIN REACTION
(1) Starting material is doubled stranded DNA
(2) Strands of the DNA are separated by heating the mixture
(3) The mix is cooled and the primers then anneal to the borders of the region of DNA to be amplified.
(4) DNA polymerase synthesizes DNA that is complementary to the template.
(5) The mixture is heated again and this is to separate the product from the template – four templates are now available for DNA replication
(6) Polymerase synthesizes DNA again, except the extension is limited to the precise section of DNA that is desired.


The following chart outlines the cycling temperatures and the length of time each step is incubated at.

BASIC PCR CYCLING PARAMETERS/PATTERNS

STEP

TEMPERATURE

LENGTH OF
TIME

 

#1) Denature DNA

92-950C

15s – 2 min

 

This completes one cycle

#2) Anneal Primers

40-700C

30-60 s

#3) Primer Extension

2-740C

1 – 2 min








The cycling parameters and reagent concentration/volumes will not always be the same for each reaction performed – these depend upon the DNA template used and reagent volumes depend upon one another.
The number of cycles will differ from reaction to reaction – the PCR process may stop due to depletion of reagents.





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Reverse Transcription



PCR replicates strands of DNA and the source comes from a DNA template or sample that contains DNA. Therefore, when RNA is extracted from a sample for duplication and study, one still employs the PCR process exactly described above – except an additional step precedes the reaction where the RNA is converted to DNA. We can specifically classify a PCR that includes this additional step as Reverse Transcription PCR.
An example of when this would happen is when you are dealing with RNA viruses – this is the case in my project since I am using nucleic material from an influenza virus (which contain RNA).





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Real-Time PCR


Now that I have covered the basics of PCR or normal/conventional PCR, we can touch upon a different type of PCR. Many types of PCR have since been developed and further classified: nested PCR, inverse PCR, reverse transcription PCR, asymmetric PCR, quantitative PCR, real-time PCR, touchdown PCR, and colony PCR, etc.
Real-time PCR uses the basic PCR reaction to amplify DNA; it has the same purpose, same basic materials and same three-step process. Real-time PCR is a type of PCR used in my project - it is carried out in a more sophisticated type of thermal cycler called a SmartCycler. The PCR reaction is run in the SmartCycler which is hooked up to a computer. With the SmartCycler and real-time PCR, we can observe the formation of PCR product as the reaction is in procession, or in “real time.” What allows the continual visualization of product formation is the additional feature of the fluorescent probes. The following is a diagram of the SmartCycler and the reaction tubes used:

Real-time PCR is exactly like that of conventional PCR except it has the additional use of probes (there are many different types, but in my project, I used the hydrolysis probe, 6-FAM). A probe is what allows us to detect fluorescence and in turn, the amplification of PCR amplicon/product. A probe consists of a reporter and a quencher. The probes are designed to anneal themselves somewhere on the sequence between the two primers (on the target region of amplification). The DNA polymerase enzyme replicates DNA and when it encounters the reporter, the polymerase will cleave or release the reporter from the probe. When the reporter is released a fluorescent signal is released. This fluorescence is directly related to the formation of products. (see diagram below)


MATERIALS
Real-time PCR requires the basic components mentioned in the basic PCR because real-time PCR is an extension of PCR but it evolves in sophistication and data collection.
1. Template DNA
2. dNTPs
3. Two Primers – a forward and reverse primer
4. DNA polymerase
5. Buffer
6. Magnesium
7. Other optional additives
8. One-step Reverse Transcription enzyme mix (If RNA is used)
9. Inhibitors (RNase inhibitors inhibit ribonucleases that may be present to degrade the RNA/DNA)
10. Thermal Cycler – the SmartCycler
11. Fluorescent probes


PROCESS
In real-time PCR, the basic PCR process occurs and the amplification of the DNA template is observed continuously at each step and second of the reaction. However, the cycling parameters may be slightly different from the basic PCR:

 REAL-TIME PCR CYCLING PARAMETERS/PATTERNS

STEP

TEMPERATURE

LENGTH OF
TIME

 

#1) (if start with RNA) Reverse Transcription

500C

30 minutes

 

#1) Denature DNA

950C

15 min

 

#2) Strand Separation

940C

1 s

This completes one cycle

#3) Anneal Primers AND Extension

600C

20 s





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Analyzing Results


After the completion of real-time PCR, the data that is collected can then be graphed into a sigmoidal graph. The basic representation of what a real-time PCR graph may look like is:


The graphs display the growth of amplicon or PCR product during the real-time reaction. At the beginning, there is little change in amplicon formation – this line of “no change” defines the baseline of the graph (very little or no product formation). An increase over this base indicates product formation – a fixed “threshold” can be set above this baseline.
The threshold line is set by the user and the value will always be the same for PCR of the same samples – the threshold is the cycle number where the fluorescent increase is logarithmic.
The Ct. values (cycle threshold) can be seen in the graph and they have also been charted above. Ct. Values are the cycle numbers where the fluorescence crosses the threshold line. Generally the greater the amount of DNA template will cause a faster encounter with the threshold (or a smaller Ct. value). Diluted samples will have increasing Ct. values since less DNA is present. Basically, the Ct indicates when the PCR product is first visualized or “formed” – a lower Ct value means that fewer cycles are required for the product to form.


The graph has three major phases:
1. Linear Phase (where the graph shows no fluorescence or very little)
2. Exponential growth phase (shows the significant growth in PCR product)
3. Plateau phase (where the graph or growth tapers off due to depletion in reagents)





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