Titiek Sunaryati
Dosen Fakultas Kedokteran Universitas Wijaya Kusuma Surabaya
Abstrak
Real-time PCR, disingkat RT PCR, adalah alat yang umum untuk mendeteksi dan menghitung ekspresi profil dari gen-gen tertentu. PCR biasanya digunakan untuk menggandakan lokasi spesifik dari suatu rantai DNA (DNA tujuan). Terdapat tiga teknik blotting yang berbeda seperti: Southern blot, northern blot, dan western blot. Southern blot untuk mendeteksi DNA, northern blot untuk mendeteksi mRNA (messenger ribonucleic acid) dan western blot untuk mendeteksi protein.
POLYMERASE CHAIN REACTION
Titiek Sunaryati
Lecturer Faculty of Medicine, University of Wijaya Kusuma Surabaya
Abstract
Real-time PCR, hereafter abbreviated RT PCR, is becoming a common tool for detecting and quantifying expression profiles of selected genes. PCR is used to amplify a specific region of a DNA strand (the DNA target). There are three different blotting techniques: Southern blot, northern blot and western blot. The Southern blot detects DNA, the northern blot detects mRNA (messenger ribonucleic acid) and the western blot detects proteins.
Introduction
The invention of polymerase chain reaction (PCR) by Kary Mullis in 1984 was considered as a revolution in science. Real-time PCR, hereafter abbreviated RT PCR, is becoming a common tool for detecting and quantifying expression profiles of selected genes. The technology to detect PCR products in real-time, i.e., during the reaction, has been available for the past 10 years, but has seen a dramatic increase in use over the past 2 years. A search using the key word real-time and PCR yielded 7 publications in 1995, 357 in 2000, and 2291 and 4398 publications in 2003 and 2005, respectively. At the time of this writing, there were 3316 publications in 2006. The overwhelming majority of the current publications in the field of the genomics have been dealing with the various aspects of the application of methods in medicine, with the search for new techniques providing higher preciosity rates and with the elucidation of the principal biochemical and biophysical processes underlying the phenotypic expression of cell regulation. Series of RT PCR machines have also been developed for routine analysis (Deepak, 2007).
PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of up to ~10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size (Cheng, 1994).
A basic PCR set up requires several components and reagents. These components include:
· DNA template that contains the DNA region (target) to be amplified.
· Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target.
· Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
· Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
· Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
· Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis.
· Monovalent cation potassium ions (Joseph, 2001).
Procedure
The PCR usually consists of a series of 20 to 40 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps. The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.
Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.
· Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
· Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
· Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.
· Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended (Chien, 1976).
The way to detect PCR results
There are three different blotting techniques: Southern blot, northern blot and western blot. The Southern blot detects DNA, the northern blot detects mRNA (messenger ribonucleic acid) and the western blot detects proteins.
The blotting techniques are often used to detect genetic abnormalities (when blotted for DNA), as well as certain infectious diseases (when blotted for protein).
a.The Southern Blot
The Southern blot is used to detect and identify certain DNA sequences in a sample of bodily fluid. It uses single-stranded DNAs to search out their complementary strands.
When a Southern blot is performed on DNA, the first step is to incubate the DNA with restriction enzymes. Restriction enzymes cut DNA at known sequences, and produces DNA fragments of a certain length. Once the DNA is cut into pieces, scientists conduct electrophoresis to separate them by size.
Then, since the DNA in the gel is double-stranded, it is separated into single strands. The single-stranded DNAs are then transferred to a special piece of paper since a labelled probe cannot reach them inside the gel (too thick). The transfer is exact and does not disturb the location and grouping of the DNAs from gel to paper. This is called "blotting."
The location is important because scientists know what a certain DNA sequence profile should look like. Since there are usually many fragments on a blot, labelled probes are used to flag the different pieces of DNA. If the specific labelled probe meets a complementary pair on the paper blot, it will bond to it. Later, a method called autoradiography is used to read the location of the attachment. Since the identity of the labelled probe is already known, the identity of the DNA from the sample will also be known.
b.The Northern Blot
The only difference between a Southern blot and a northern blot is that the northern blot uses mRNA from samples instead of DNA. So instead of applying gel electrophoresis to DNA, northern blot applies it to mRNA, and since mRNA is naturally single-stranded, there is no need to separate the strands as in a Southern blot.
c.The Western Blot
The western blot is used to detect different proteins. Gel electrophoresis is applied to the specific proteins. This separates them by size and just like in the other blots, the result is blotted onto a special paper. The rest of the steps follow the method for an ELISA (Brown, 2001).
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