Structural Biochemistry/DNA Amplification Technique:Polymerase Chain Reaction (PCR)

History

In 1984, Kary Mullis invented a method to amplify DNA sequences. In previous attempts, DNA polymerase cannot withstand the temperature change. However, Mullis used polymerase from Thermus aquaticus, a bacteria that lives in hot springs in his method, which allowed PCR to be performed at high temperature conditions.

Overview

Polymerase Chain Reaction (PCR) is a very effective technique of obtaining multiple identical copies of a certain DNA strand (amplifying DNA). PCR can be used for amplifying DNA, mutation DNA, delete DNA, and introduce restriction endonuclease site. PCR is performed by repeating a cycle that consists of several steps. This process is performed after regions of target DNA, or the sequence(s) of DNA which are to be amplified (Target Sequence), have been specified. Target Sequence, as its name implies, is a specific part of DNA that is needed for further study. In short, one cycle of PCR consists of these steps:

Preparation before repetition of PCR cycle:
- Addition of PCR components to solution containing DNA to be amplified
  - This step is only performed before the first PCR cycle. Addition of components after the first cycle is not advised because PCR reaction is temperature sensitive (slight change in temperature can greatly affect the reaction)
  - Components to be added are as followed:
    - DNA template to be amplify during this process.
    - A pair of primers (usually between 10 - 30 nucleotides long) that is complementary to the Flanking Sequence (several bases directly left or right of the target sequence). These two different primers will be referred as primer F (for the forward primer) and primer R (for the reverse primer).
    - The four dNTPs (deoxyribonucleotide triphosphates); they are: dATP, dGTP, dCTP, and TTP
    - Magnesium Chloride (MgCl₂) in order to stabilize the charge of phosphate group and activate the replication process.
    - Heat stable DNA Polymerase; heat stability is very important because PCR reaction is performed at various temperatures. This heat stable DNA polymerase is obtained from a thermophilic bacteria Thermus aquaticus, the inhabitant of hot springs. Hence, this DNA polymerase is termed as Taq DNA Polymerase. Other polymerases with different properties can be used depending on the purpose and requirements of the PCR; Some polymerases, such as Pfu Turbo, for example, have higher fidelity rates but are much more sensitive.
    - Buffer solution to stabilize the DNA sample.
    - To maximize the efficiency of this process, a master mix is made and contained of the primer, MgCl₂, buffer, and Taq polymerase.
  - These component must be added in excess to ensure that when the two DNA strands are separated in the second step, the chance of the two DNA to re-hybridize is minimized.

PCR cycle (perform about 20-30 cycles):
1. Strand Separation
  - Strand separation is performed to expose target sequence and flanking sequence to primer. This step is done by heating the solution, causing the hydrogen bonds maintaining the double helix structure to break.
  - Parameter:
    - Temperature: about 95^oC
    - Time: 15 to 30 seconds (to separate the DNA to 2 single strands)
2. Hybridization of Primers (Also known as the annealing step)
  - At this step, the solution is cooled to around 54^oC. By cooling down the solution, primers are allowed to hybridize with the flanking sequences via hydrogen bonding.
  - It is very possible that the two main strand will re-hybridize because both strands are complementary to each other. Fortunately, as explained earlier, it can be easily avoided by having excess primers in solution. Excess primers are expected to hybridize with the flanking sequence before any two complementary DNA can hybridize.
3. DNA Synthesis (aka elongation step)
  - Solution heated to 72^oC. At this optimal temperature, the Taq polymerase will start elongates both primers.
  - There are two types of primer; each is the complementary oligonucleotides of the flanking sequence.
  - Elongation happens not unlike the regular polymerization of DNA. The primers are elongated from the 5' to the 3' ends (which is the opposite direction of the actual DNA strand; strand that have target sequence, flanking sequence, and un-amplified sequence).

- Note:
  - By the end of first cycle, we obtain 2 types of new strand in addition to the original DNA strands. These strands are the elongation of each type of primer. They contain:
    - One of the primer (exp: primer A)
    - The target sequence
    - A complementary of a flanking sequence (the one that is not hybridized to the primer contained in this strand; e.g.: complementary of flanking sequence of primer B)
    - The rest of the non-target sequence after this complementary of flanking sequence.
  - On the second cycle, some of the primers will hybridize with the new type of sequence obtained on first cycle (exp: on the shorter strand elongated from primer A, primer B will attach to its flanking sequence and start elongates the cycle until it reaches the end of the shorter strand, the flanking sequence of primer A).
  - On the second cycle, the new type of strand formed, Short Strand consist of:
    - One of the primer (exp: Primer B)
    - The target sequence
    - The flanking sequence that can hybridize with Primer A
  - On the second cycle, besides forming the short strand, the same strands as the one formed on the first cycle can also form.
  - On the second to the n^th cycle, the short strand is amplified exponentially while the slightly longer strand (the only strand that form on the first cycle) is amplified arithmetically.
  - This cycle is repeated n times. At the n^th repetition, there will be 2ⁿ of desired sequence.

File:PCR Steps.jpg

PCR process with detailed steps.Can be repeated in multiple cycle to amplify original DNA structure

Finally, the reaction is usually held at 4 degrees Celsius after completion to stabilize the DNA, which is temperature sensitive.

Additional Steps

A final elongation step is performed at 70-74°C for at least 5 minutes after the last PCR cycle has been done. This step will guarantee that any remaining DNA strand is fully extended. In addition, a hold step at 4°C for an indefinite time can be utilized for storage of the DNA product.

Thermal Cyclers

File:New PCR machine.jpg

PCR machine with a temperature gradient

The thermal cycler can perform different steps of PCR process by cooling and heating the tubes constantly. They use of the Peltier effect which allows both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes with no RNAase and DNAase are suitable for PCR. Due to their thickness, they can create thermal conductivity to allow for rapid thermal equilibration. As showed in the next picture, most modern thermal cyclers will have heated lids to prevent condensation at the top of the reaction tube and evaporation in the bottom of each tube.

Results Interpretation

To ensure the quality of PCR amplification, a gel-electrophoresis is performed. To prepare for this step, a series of steps need to be done before hands

1. Preparation 1% agarose gel: Weight out 1 gram of purified agarose and dissolve to 100 ml with TAE (Tris-Acetate) buffer solution. (Figure A)

**Figure A**: Agarose powder (right) and ethidium bromide (left, brown bottle)

2. Heat up. The solution is heated up in microwave until all of agarose particles dissolved completely.

3. Addition of ethidium bromide. After the solution is cooled down, 5 microliters of ethidium bromide are added to the solution. Ethidium Bromide will attached to DNA molecules and make the strand stand out in UV light background (used as a nucleic acid stain).

4. Polymerization of agarose. Wait for the agarose to completely polymerize after inserting a well plate to load the samples into. This process can take up to 30 minutes.

**Figure B:** Polymerization of agarose. The process can take up to 30 minutes. During this process, the tray cannot be moved

5. Addition of buffer solution. Fill up the chamber with TAE solution to create a barrier between the outside environment and the DNA sample.

6. Addition of DNA sample. Loading dye should be added to the DNA samples, and an appropriate DNA ladder run in order to determine the approximate molecular weight of the sample in kilobases.

7. Apply electrical current. The DNA must be located so it will near the negative charged node and away from the positive node.

The electric current is applied to the chamber. The top node is connected to the negative node while the bottom part is connected to the positive node.

**Figure E:** A voltage machine is in process

8. Visualize the results using UV light.

File:UVVisualization.jpg

Figure F: UV Visualization of gel

Advantages

PCR is a popular method in amplifying DNA because it brings various benefit. Some of those benefits are:

Researcher does not need to elucidate the sequence of the target sequence. They only need to know the sequence of the flanking sequence.
Target that are much larger than the primer can still be synthesized.
Primers prepared does not have to be an exact match of the flanking sequence.
PCR is very specific.
PCR is very sensitive to the point that it is able to amplify a single DNA molecule.
The resultant DNA can be processed further more such as mutation, deletion, or cloning process

Mechanism

In this example, the following convention is used:

BOLD = flanking sequence and its complementary sequence
ITALICS = target sequence and its complementary sequence
NORMAL = the remaining sequence (the non amplified part of DNA strand)
BOLD/ITALICS= primers

Sequences formed at various stages of PCR:

Original DNA molecule:

...CGCG TTG CAGCTACTGACTC CCC CGCG...
...GCGC AAC GTCGATGACTGAG GGG GCGC...

After separation and attachment of primer (beginning of first cycle):

...CGCG TTG CAGCTACTGACTC CCC CGCG...

AAC

CCC

...GCGC AAC GTCGATGACTGAG GGG GCGC...

After first sequence elongation (end of first cycle):

...CGCG TTG CAGCTACTGACTC CCC CGCG...

AAC GTCGATGACTGAG GGG GCGC...

...CGCG TTG CAGCTACTGACTC CCC
...GCGC AAC GTCGATGACTGAG GGG GCGC...

After separation of sequence and attachment of the other primer (beginning of second cycle; the formation of sequence identical to sequence formed in first cycle is not shown):

...CGCG TTG CAGCTACTGACTC CCC CGCG...

AAC GTCGATGACTGAG GGG GCGC...

CCC

AAC

...CGCG TTG CAGCTACTGACTC CCC

...GCGC AAC GTCGATGACTGAG CGG GCGC...

After sequence elongation (formation of Short Sequence, end of second cycle):

...CGCG TTG CAGCTACTGACTC CCC CGCG...

AAC GTCGATGACTGAG GGG GCGC...

TTG CAGCTACTGACTC CCC

AAC GTCGATGACTGAG GGG

...CGCG TTG CAGCTACTGACTC CCC

...GCGC AAC GTCGATGACTGAG CGG GCGC...

After separation of short sequence and addition of primers (beginning of third and subsequent cycle; only the short sequence formation is shown):

...CGCG TTG CAGCTACTGACTC CCC CGCG...

AAC GTCGATGACTGAG GGG GCGC...

TTG CAGCTACTGACTC CCC

AAC

CCC

AAC GTCGATGACTGAG GGG

...CGCG TTG CAGCTACTGACTC CCC

...GCGC AAC GTCGATGACTGAG CGG GCGC...

After elongation of short sequence (end of third and subsequent cycle; only the short sequence formation is shown):

...CGCG TTG CAGCTACTGACTC CCC CGCG...

AAC GTCGATGACTGAG GGG GCGC...

TTG CAGCTACTGACTC CCC

AAC GTCGATGACTGAG GGG

TTG CAGCTACTGACTC CCC

AAC GTCGATGACTGAG GGG

...CGCG TTG CAGCTACTGACTC CCC

...GCGC AAC GTCGATGACTGAG CGG GCGC...

Short sequences will be re-separated so that new primers can attach in the subsequent cycle:

TTG CAGCTACTGACTC CCC

AAC

CCC

AAC GTCGATGACTGAG GGG

TTG CAGCTACTGACTC CCC

AAC

CCC

AAC GTCGATGACTGAG GGG

Primers will be elongated to form additional short sequences. These last two bullet points are repeated on subsequent cycle until sufficient amount of target sequence has been synthesized:

TTG CAGCTACTGACTC CCC

AAC GTCGATGACTGAG GGG

TTG CAGCTACTGACTC CCC

AAC GTCGATGACTGAG GGG

TTG CAGCTACTGACTC CCC

AAC GTCGATGACTGAG GGG

TTG CAGCTACTGACTC CCC

AAC GTCGATGACTGAG GGG

One More PCR Diagram

Media:PCR diagrams.pdf

Practical Applications of PCR

Polymerase chain reaction has become an important tool for medical diagnosis. PCR can detect and identify bacteria and viruses that cause infections such as Tuberculosis, Chlamydia, viral meningitis, viral hepatitis, HIV and many others. Once primers are designed for the DNA of a specific organism, using PCR to detect the presence or absence of a pathogen in a patient’s blood or tissue is a simple experiment. Because PCR can easily distinguish among the tiny variations in DNA that each of us posses and that make each of us genetically unique, the method is also leading to new kinds of genetic testing. These tests diagnose not only people with inherited disorders but also people who carry deleterious variations (mutations) that could be passed on to their children. These carriers are usually not themselves affected by the mutant gene but they can lead to a disease in the next generations (e.g. mutations that cause cystic fibrosis). Many of the new genetic tests are the result of the Human Genome Project, the huge international effort to identify and study all human genes. The project is progressing rapidly towards its ultimate goal which is to sequence the entire DNA in typical human cells. Sequencing DNA means to determine the precise order of the four different nucleotides that make up any strand of DNA. DNA sequencing reveals crucial variations in the nucleotides that constitute genes. These mutational changes produce diseases and even death by forcing the genes to produce abnormal proteins or sometimes no protein at all. DNA sequencing involves first isolating and duplicating DNA segment for nucleotide analysis. Thus PCR is an essential tool for the Human Genome Project because it can quickly and easily generate an unlimited amount of any piece of DNA for this kind of study. PCR is a direct way of distinguishing among the confusion of different mutations in a single gene, each of which can lead to a disorder such as Duchenne Muscular Dystrophy. It also helps doctors track the presence or absence of DNA abnormalities characteristic of a particular cancer so that they can start and stop drug treatment and radiation therapy as soon as possible. PCR promises to greatly improve the genetic matching of donors and recipients for bone marrow transplantation. The technique incomparable ability to identify and copy the smallest amount of even old and damaged DNA has proved exceptionally valuable in the law, especially the criminal law. PCR is an indispensable adjunct to forensic DNA typing- commonly called DNA fingerprinting. Traces of DNA found at a crime scene can also be amplified by using PCR, thereby providing sufficient amount of DNA to match with the suspect’s DNA. DNA is extremely stable molecule, especially when protected from air, light, and water. Under these circumstances, large pieces of DNA can be preserved for thousands of years or longer. Thus PCR provides an excellent method for amplifying such ancient DNA molecules, so that they can be subjected for analysis. This technique can also be used in amplifying DNA from microorganisms that have not yet been isolated and cultured. Sequences from these PCR products can be sources of considerable insight into evolutionary relationships between organisms.

Variations of PCR

Because of the high adaptability of PCR, many new biochemical techniques based on the original PCR method have been invented. Some of the more commonly seen ones are listed below in alphabetical order.

Allele-specific PCR is a cloning technique based on single-nucleotide polymorphism (SNPs). This requires a DNA sequence that has differences between alleles and uses primers whose 3' end encompass the SNP.

Assembly PCR, more formally known as Polymerase Cycling Assembly (PCA), is a method of synthesizing long strands of DNA oligonucleotides by applying PCR to small oligonucleotides and then hybridizing them together. The entire process consists of two sets of PCR reactions. The first reaction utilizes forward and reverse primers to anneal together oligonucleotide fragments with complementary sequences. The second reaction uses another set of primers to perform a regular PCR reaction, which amplifies the results of the first reaction. Only the larger oligonucleotide strands are amplified, distinguishing them from the other incomplete shorter fragments. Once the entire reaction is performed, gel electrophoresis may be performed to isolate and identify the completed strands.

Asymmetric PCR is a biochemical technique that focuses on the amplification of one strand of DNA over the other. To do so, a normal PCR reaction is performed but with either an excess of primers that react to the target strand or absence of primers that react to the complementary strand. This method is utilized in certain types of forensic sequencing and probing, where having only one of the two DNA strands is preferred.

Colony PCR is a biochemical technique of amplifying DNA in a vector, such as E. Coli bacterial colonies, to identify the target DNA. The first step in the process involves removing a sample of the vector from the growth plate by a sterile toothpick or pipette tip. The sample is transferred to either a PCR master mix or autoclaved water, where a normal PCR reaction is performed to determine whether or not the colony contains the DNA fragment or plasmid of interest.

Digital PCR simultaneously amplifies thousands of samples.

Helicase-dependent amplification uses constant temperature instead of cycling through denaturation, annealing, and elongation cycles. DNA helicase, an enzyme, unwinds DNA and used to replace thermal denaturation.

Hot-start PCR is a form of PCR that focuses on the maximization of product yield. To reach this goal, chemical modifications are used to limit polymerase activity prior to the reaction. This limitation reduces non-specific polymerase amplification. Next, the reaction, itself, is performed at extremely high temperatures using polymerase obtained from thermophilic organisms, such as Achaea bacteria from ocean vents. These elevated temperatures further prevent non-specific polymerase amplification, resulting in a larger yield of the target product.

Intersequence-specific PCR a method for DNA fingerprinting. this amplify regions to produce a unique fingerprint of amplified fragments.

Inverse PCR is a method engineered to bypass the limitations of regular PCR, which requires prior knowledge of the flanking sequences around the target sequence. Inverse PCR is aptly named because it can be performed to determine the flanking sequences of a target strand. However, this method has its own limitation: one internal sequence must be known for it to be performed.

Ligation-mediated PCR is a method used for DNA sequencing, genome walking, DNA footprinting. This uses small DNA linkers ligated to desired DNA and multiple primers annealing to the linkers.

Methylation-specific PCR (MSP) detects methylation of CpG islands in genomic DNA. DNA is treated with sodium bisulfate and is recognized by PCR primers. Two PCRs are used to modify the DNA. One primer recognizes DNA with cytosines to amplify methylated DNA, and the other primer recognizes DNA with uracil/thymine to amplify unmethylated DNA.

Miniprimer PCR allows PCR targeting to smaller primer (smalligos) (consists of 9-10 nucleotides) binding regions and used to amplify conserved DNA sequences.

Multiplex PCR is a biochemical technique that focuses on simultaneous amplification of multiple target DNA products through the addition of multiple primers into a single PCR reaction. Multiplex PCR is a preferred method for DNA testing because it allows for the analysis of deletions, mutations, and polymorphisms in a sample.

Multiplex Ligation-dependent Probe Amplification (MLPA) amplifies multiple targets with only a single pair of primer.

Nested PCR is a form of PCR that maximizes accuracy in the amplification of a target DNA fragment. Nested PCR consists of two separate reactions. The first is a normal PCR reaction where a pair of primers is used to amplify a target fragment. The second reaction adds a second pair of primers called “nested primers” that bind to the products of the first reaction and amplify them. By performing two sets of PCR reactions, this method maximizes accuracy since it is very unlikely that two pairs of primers would both bind to the wrong target fragment.

Overlap-extension PCR allows the construction of DNA sequence with an alteration inserted beyond the limit of the primer length.

PAN-AC uses isothermal conditions for amplification.

Quantitative PCR (Q-PCR) measures the quantity of a PCR product. It measures the starting amount of DNA, cDNA, and RNA. this is commonly used to determine if a dNA sequence is present and the number of its copies in the sample. This uses fluorescent dyes, for example TaqMan, to measure the amount of amplified product.

Reverse Transcription PCR (RT-PCR) is a biochemical technique engineered to make good use of our knowledge of how reverse transcription can form complementary DNA from RNA. RT-PCR is performed in two major steps. In the first step, reverse transcription of a target RNA strand into its complementary DNA is performed using oligo-DT, which substitutes for the role of primers. In the second step, a regular PCR reaction is performed by adding primers specific to the DNA to help amplify the target sequence. RT-PCR is a highly sensitive technique that takes advantage of the smaller size of RNA strands (when compared to their DNA counterparts). RT-PCR is a highly applicable technique. In addition to the aforementioned material, this technique can be used for the mass production of target RNA through the synthesis of complementary DNA, which can be subsequently cloned through a vector.

Touchdown PCR is a PCR technique that focuses on maximizing target product yield. In such a way, it can considered an alternative to the Hot-start PCR method. To achieve its goal, this method performs PCR reactions at high annealing temperatures, which reduces non-specific polymerase amplification. In other words, only the fragments containing the sequences of interest will be amplified by the primers. As the fragments are amplified over and over again, the temperature is gradually lowered until it is certain that only target fragments have remained.

Universal Fast Walking is used for genome walking and genetic fingerprinting. This is a more specific two sided PCR than the normal one sided PCR approach.

Variable Number of Tandem Repeats (VNTR) PCR target areas of the genome that exhibit length variation. The analysis of the genotypes involves sizing of the amplification products by gel electrophoresis. Analyzing smaller VNTR segments (short Tandem Repeats, STRs) is the base for DNA Fingerprinting.

DNA Polymerases

Klenow fragment is originally derived from the DNA Polymerase I from E. coli (first enzyme to be used in PCR). This polymerase needs to be replenished every cycle due to its lack of stability at high temperature. This is not commonly used in PCR.

Bacteriophage T4 DNA polymerase was initially used in PCR but is also destroyed by heat and has a higher fidelity replication than the Klenow fragment.

Thermus aquaticus (Taq) is commonly used and is the first thermostable (heat-stable) polymerase used in PCR. This enzyme is isolated from a native source or from its cloned gene expressed in E. coli.

Stoffel fragment is made of a truncated gene for Taq polymerase and expressed in E. coli. This enzyme lacks a 5’-3’ exonuclease activity and is able to amplify targets longer than its native enzyme.

Faststart polymerase is a variant of Taq polymerase that requires a strong heat activation. This should be avoid from non-specific amplification because of its low temperature polymerase activity.

Pfu DNA polymerase is isolated from an archean Pyrococcus furiosus. This has a proofreading activity. Since errors increase as PCR progresses, Pfu is preferred when the products are individually cloned for sequencing or expression.

Vent polymerase is extremely thermostable and is isolated from Thermococcus litoralis.

Tth polymerase is a thermostable polymerase from Thermus thermophilus. This polymerase has a reverse transcriptase activity when in presence of Mn2+ ion that allows PCR amplification from RNA targets.

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