Week 14 posting

Hey all. I sincerely apologise for the late posting as I was confused over the dates and the weeks. Anyway, I’ll continue from where I left of the previous post that is gel check for primer optimization. After the gel check for primer optimization and the optimum conditions have been chosen for the specific primer, PCR can then be done for that primer.

What are the differences and similarities between primer optimization and PCR?

Differences

-Primer optimization is done using test DNA whereas PCR is done using a specific patient’s (whether healthy or diseased) DNA. This is because, when doing the primer optimization, the optimum conditions are not known, thus, it is not necessary to use patient’s DNA. (Patient’s DNA is very precious as they are retrieved only ONCE from the patient themselves while test DNA can be bought.)
-For primer optimization, a range of temperatures will be programmed at the Thermogradient, whereas, for PCR, a specific optimum temperature will be set for the Thermocycler.

Similarities
- The method for conducting both PCR and primer optimization is similar including all the reagent used.

Introduction on PCR

Polymerase Chain Reaction (PCR) is a method for the amplification of specific DNA sequences in vitro, which involves repetitive heating and cooling cycles to yield large quantities of replicated DNA. It requires two oligonucleotide primers that will flank the DNA target sequence that is to be replicated. The three major steps that are carried out in each PCR cycle are the following:
1. Denaturation: The hydrogen bonding that holds the double stranded of the target DNA molecule together, are separated into single stranded DNA by heating at 940C.
2. Annealing: The two single stranded DNA are then allowed to cool, at the temperature ranging from 45oC to 60oC, where annealing of the primers to the single stranded DNA will take place. This would initiate the synthesis of the complementary sequences.
3. Extension: When the temperature is increased to a temperature optimum for the Taq DNA polymerase, it will bind to the free 3’ end of the primers. By incorporating dNTPs, the Taq DNA polymerase synthesizes a new DNA strand in a 5’ to 3’ direction.

Method for PCR
*Please refer to previous entries on method for primer optimization as they are similar*

After which, gel check has to be done again. This time, it is to double check if the bands are present for different DNA samples. However, for the gel check of PCR, only 0.5ul of loading dye and 2ul of random PCR product are used instead of 2ul of loading dye and 10ul of primer optimization product. This is because, the remaining 8ul of the PCR product is required to be used for experiments following this step. If approximately 80% of the bands appear, purification of the PCR product can be done.

Purification

To eliminate excess PCR components after the PCR reaction, purification of the amplified PCR products was done using two hydrolytic enzymes, Exonuclease 1 (Exo 1) and Shrimp Alkaline Phosphatase (SAP). Exonuclease 1 catalyzes the removal of excess primers in the 3’ to 5’ direction while Shrimp Alkaline Phosphatase is responsible for the dephosphorylation of dNTPs to prevent it from participating in any further polymerisation steps. Both enzymes at its optimum temperature, 37oC, are involved in the digestion process. Following this, the temperature will increase to 72oC resulting in the denaturation and inactivation of the enzymes. This would prevent the enzymes from interfering in subsequent steps, for example, cycle sequencing.

Methods for purification

Following PCR and gel electrophoresis, 0.5µl of Exonuclease 1 (Exo1) and 1µl of Shrimp Alkaline Phosphatase (SAP) need to be added into each sample of the PCR products.
1. Calculations were done by multiplying the number of samples needed to be purified.
2. One 1.5ml of eppendorf tube (master mix tube) was collected and labeled ‘Exo-Sap’.
3. An ice box, filled with crushed ice, was prepared.
4. Exo 1 and SAP was retrieved from -20oC fridge and placed in the ice box.
5. The desired amount of Exo and SAP was pipetted into the master mix tube.
6. The master mix tube was then spun down using a mini centrifuge.
7. 1.5ul of the resultant mixture was pipetted into each sample in each well.
8. The samples of the PCR products were placed into the thermocycler and the program was set.

After purification is done, the next step would be sequencing. I would explain this step in further details in the next posting. Till then, I would like to wish all Muslims, Selamat Hari Raya Aidilfitri and to all non-Muslims, have a joyous holiday! J

Liyanah Zaffre
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