PCR-RFLP Report
PCR
What is it: The Polymerase Chain Reaction is a method that uses the capability of DNA polymerase to synthesize to new DNA strands which are matching to the template strand. A primer needs to be added to the first nucleotide due to the fact that DNA polymerase only can add a nucleotide only onto a 3 '-OH group that already exists. Because of this condition, we are able to define a chosen region of template sequence which we can then generate millions to billions of copies. This technique was developed by Kary Mullis in 1983 and is a very common indispensable technique which has a variety of uses such as DNA cloning for sequencing, genetic fingerprints, detection and diagnosis of infectious disease (often cancer) etc...
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To achieve this, the reaction must be heated to 94-98 Degrees Celsius for about 20 to 30 seconds.
Annealing Steps: Annealing must take place in order for the hybridization of primers to the single stranded DNA template. This temperature must be high enough in order for the hybridizations to bind specifically and low enough that it is possible for hybridization of primers to occur. Annealing temperature is usually 3-5 Degrees Celsius lower than the melting temperature of the primers.
Extension/elongation step: This step consists of DNA polymerase synthesizing with a new DNA strand which is complementary to the DNA template strand. This is done by adding dNTP 's which complement the template in the 5 '-3 ' strand, condensing the 5 '-Phosphate group of the dNTP along with the 3 '-hydroxyl group at the end of the nascent extending DNA strand. The temperature at which the reaction is treated depends upon the primer used. The duration of this step also depends on the DNA polymerase used also, and the length of DNA fragment used. Under optimal required temperature of the primer, the DNA polymerase will polymerize a thousand base pairs per minute.
Final elongation: After the last PCR cycle is completed, the reaction is cooled to a temperature of 70-74 Degrees Celsius (optimal temperature needed for activity for most polymerases
20 ul of DNA was added to 20ul of Master Mix. The Master Mix contained primers, dNTPs, Mg2+, Taq DNA polymerase, and yellow dye. Both the DNA and Master Mix were mixed with the micropipette. The DNA was then put into the thermal cycler containing 40 cycles of PCR amplification, amounting to 3.5 hours of amplification.
Instead, a PNA strand invaded the DNA helix , displacing one of the DNA strands to form a bond with its (PNA) complement, and the second PNA strand formed what is called a Hoogsteen bonds PNA-DNA= PNA triplex. This created a triple invasion of the DNA helix. Other bonding methods were experimented where the PNA’s bases where modified according to the targeted DNA. Fortunately this method had better results though there are a lot more things to overcome.
Use a test tube holder to put the test tube into a container of boiling water for 5 minutes, or until the solution changes color.
Requirments: The total reaction volume was 10 μL, containing 1 μL of 10× LightCycler DNA Master Hybridization enzyme mixture (Roche Diagnostics), 3 mM MgCl2, 0.1 μM hybridization probes, and ARMS primers.
After that, a new sterile loop was used to immerse the pGLO plasmid DNA stock tube into the +pGLO tube but no into the – pGLO tube. Both tubes were well closed and put back on the ice for 10 minutes. While both tubes were sitting on ice, the four plates were labeled. LB/amp plate was labeled +pGLO, LB/amp/ara plates were labeled +pGLO, LB/amp plate was labeled – pGLO, and LB plate was labeled – pGLO. After that, the tubes were transferred into a water bath set at 42° C for exactly 50 seconds, then placed rapidly back on the ice for another two
In preparing for the bacterial transformation, DNA plasmid is introduced into the E. coli cells that will express newly acquired genes. Two tubes were used and labeled both as +pGLO and -pGLO. A solution of (CaCl2) was transferred 250 µl onto the two tubes. The tubes were placed on the ice. A sterile loop was then used to gather a single colony of bacteria from a starter plate. Now, that both tubes contain bacteria they were placed on the ice for 10 minutes. Four agar plates were labeled as: +pGLO LB/amp, +pGLO LB/amp/ara, +pGLO LB, -PGLO LB/amp. Heat shock was used to transfer both the +pGLO and -pGLO, at exactly 42°C. Time was observed for 50 seconds and quickly return the tubes to the ice for another 2 minutes. As the tubes, cold down they
Since the temperature optimum for the DNA polymerase to act is 72° C, the reaction is heated to that temperature. DNA polymerase lengthen the primers by attaching more nucleotides onto the primer in a sequential manner, using the target DNA as a template.
To start this laboratory, it must first label two transformation tubes of different color; one with – DNA and the other with + DNA. Then, using a P-1000 micropipette adds 250 ul of the ice cold transformation solution (CaCl2) into each tube and places both tubes on ice. Using a disposable sterile inoculating loop to transfer colonies of E. coli bacteria from the starter plate to the +DNA tube. Immerse the loop in the CaCl2 solution in the transformation tube and spin the loop until all bacteria is incorporated into the transformation solution. Repeat this step with the – DNA tube. Using a P-10 pipette, transfer 10 ul of pGLO directly into the + DNA tube. Tap the tube lightly with a finger to mix. Place both tubes onto ice, and incubate for 10 minutes.
The primary purpose is to identify a genetic marker or study the function of a specific gene. There are three steps involved in this process which are as follows: denaturation, annealing and elongation. Denaturation involves heating the DNA to agitate the hydrogen bonds, and annealing allows the temperature to be lowered so that the primers can be “annealed” to the single-stranded DNA template. The last step requires DNA polymerase to synthesize a new strand of DNA that is complementary to the RNA strand in the 5’ to 3’ direction (Amplifying DNA: The Polymerase Chain Reaction, 2016). The forward and reverse primers are needed to start the replication process by providing the appropriate nucleotides to the new strand. On the contrary, sanger sequencing makes copies of a target DNA, and the the DNA strand that will be sequenced is separated into two strands, so they can be copied through chemically altered bases. The altered bases cause the process of copying to terminate each time a particular letter is added to the growing DNA chain, which happens to all four bases until the fragments are put together to reveal the original sequence of the original DNA. The aforementioned processes are thoroughly explained to give an overview of the steps involved in providing the end products of the experiment, so an individual can manually decipher
PCR works by denaturing the double-stranded DNA and annealing the primers to the newly-made single-stranded DNA, leading to the extension/elongation of the DNA by a polymerase that attaches to the primer/DNA strand. The PCR reaction strums through a handful of temperature cycles to maximize each step and the amount of product.
Polymerase α is the first mammalian polymerase detected. It is a member of the B-family of replicative DNA polymerases. Its primary role is to further extend the RNA primer by addition of deoxy-nucleotides (dNTPs) into a total of 20-25 nucleotides of RNA-DNA primer. Generally, replicative DNA polymerases, polymerize dNTPs by catalyzing nucleophilic attack of the 3’-hydroxyl group of the primer onto the α-phosphate of an incoming dNTP aligned to the template strand. The accuracy of the polymerases is achieved by a confirmation of the correct base pairing of the incoming dNTP and the template strand according to Watson-Crick base-pairing. The accuracy of the primases is further enhanced (100 fold) by their exonuclease activity where in the event of a mismatch the growing strand is pushed back to the exonuclease active and the mismatched nucleotide is excised.
The second stage of the process is complementary base pairing. In this stage, new complementary nucleotides are positioned following the rules of complementary base pairing: adenine (A) to thymine (T) and guanine (G) to cytosine (C). Then, the binding of free nucleotide with complementary bases is catalyzed by DNA polymerase.
The first is to denature dsDNA through heating to ~96 °C. This separates the two strands of DNA. The exact temperature to be used can be calculated with Tm = 4oC x (no. of G & C) + 2oC x (no. of A & T). Tm is the melting point of the strands and to supply the number of G, C, A, & T ‘s the primer is used.
PCR works in 3 major processes. These three steps are done repeatedly every 30-40 cycles. The 3 processes i.e. Denaturation, Annealing and Extension are all done under specific temperature gradients.
The enzymatic or the molecular “xeroxing” process in which a particular region of DNA is replicated several times to yield thousands of copies of a specific sequence is termed as Polymerase chain reaction (PCR). The process of PCR employs a sophisticated and precise pattern of heating and cooling of samples in a thermal cycler, over 30 cycles.