Week 4 Attachment Sharing

Greeting to all fellow BMT course mates!! It is time for me to share my experience in SIP with you all. Let begin!!

Basically, I am attached to a research lab, which revolves around gene expression and its relation to certain disease. Concurrently, I am working on a particular gene, which might be related to diabetes. In this research, most of time I am dealing with a lot of MBIO and MCT stuffs, for instance things like PCR, real-time PCR, cell culturing and DNA sequencing.

Introduction to real-time PCR
Real-time PCR also refers to kinetic polymerase chain reaction. It is a unqiue technique commonly used to amplify and at the same time qualify the amount of a specific DNA of choice in the sample. In fact, it detects the amount of target DNA based on the amount of fluorescence release from its probes during the PCR process.

Principles of real-time PCR
Let recall our biochemistry and molecular biology knowledge! As we have learnt in lessons, PCR amplified double- stranded DNA by adding Taq DNA ploymerase, dNTPs, DNA templates, PCR buffer, last but not least the primers (both forward and reverse). Hence, when these solutions come together in PCR, the primers will anneal to the flanking sequence of the denatured DNA. Followed by extension by Taq DNA polymerase with the aid of dNTPs.

However, in the case of real-time PCR, probes are also added into the PCR master mix solution. These probes comprised of a reporter and quencher dye each at both ends. In addition, these probes sequences are complementary to the target DNA sequences. Therefore if the target DNA is present in the sample these probes will bind to it specifically and form a hybrid. However, the 5’ to 3’ nucleolytic activity of the Taq DNA polymerase will cause the hybrid (probe- target DNA) to cleave in between the reporter and quencher dye. A new extended strand of DNA will then displace the fragmented probes as the ploymerisation continues. Since the 3’ end of the probes is blocked, there will not be any amplifying of probe. The results is then read and recorded by software. The higher the fluorescence intensity indicated a higher amount of target DNA and vice-versa.




Designing the probes
Unlike other labs, my lab synthesizes our own probes. In order to synthesizes a probe there are certain guidelines to follow, such as
· GC contents of the probe must be within 20-80% range
· Identical nucleotide must be avoid
· Avoid a G nucleotide at the 5’ end
· Choose probes that have Cs instead of Gs
· The melting temperature should maintain at 68- 70 °C
· Probe sequence must be near to primer but not overlapping it


TaqMan Allelic Discrimination Kit
TaqMan Allelic Discrimination Kit is one of the most important kits I used during my research. It employed the principles used in the real-time PCR; only different is that it consists of two types of different probes instead of one. The two type of fluorescence probes used is TET (6-carboxy, 4,7,2,7’- tetrachlorofluorecein) while the other is FAM (6-carboxy fluorecein). These two probes is covalently linked to the 5’ end of the probes. Both type of probes will bind to their respective target DNA template (in this situation is two alleles) and form a hybrid. Fluorescence signals given by the reporter will only be detected when the DNA template is polymerized.

Take example; TET represents allele 1 and FAM represent allele 2. If a sample DNA template undergoes real-time PCR and high TET fluorescence signal is detected. This could mean that the individual is a homozygous for allele 1 and vice – versa for allele. However, what if both the signals are equally high?? Hmm…


Ans: That would indicate that the individual is a heterozygous (having both allele 1 and 2) (have you got it right?? Hehe)

Procedure for real-time PCR
1. Prepared the Real-time PCR master mix by adding the following:

· dNTPs,
· PCR buffer
· Probe
· RNase free water

2. Vortex the solution briefly
3. Add in the Taq DNA ploymerase
4. Inverted the centrifuge tube 2 to 3 times to mix the solution
5. Aliquot 9ml(for my lab) into each well in the 96 wells reaction plate/ MicroAmp optical tube
6. Close the plate tightly with the MicroAmp optical caps
7. Briefly centrifuge the plate (this is to allow the droplets cling on the side to settle)
8. Place the plate onto the thermal cycler block
9. Ensure that the real-time PCR cycling is in this manner:

· 25 ºC for 10 mins
· 48 º C for 30 mins
· 95 º C for 5 mins
10. Remove the plate and stored in fridge

Tips for real-time PCR
Here are some tips to reduce the chances of getting a false positive result. High DNA concentration from sample, amplicon carryover from previous PCR or positive control can lead to false positive results. In such case, AmpErase UNG (uracil N-glycoslase) treatment can be applied. This is because when UNG (uracil N-glycoslase) solution is added, the dTTPs are substitute by dUTPs in the PCR process. Hence it prevents the reamplification of the carryover in subsequent process by degrading the misprimer or non-specific PCR products via enzymatic reaction.

Another useful tip for PCR process is the addition of Q solution. Q solution changes the melting temperature of the DNA template during the PCR process. Therefore increase the specificity of the primers anneal to the DNA template during the PCR process. Besides that, it also increases the chances of achieving successful amplification and more PCR products.

By the way, if you have any question feel free to give me your comments.
Till then enjoy your SIP and see you all in the upcoming week!!

Avery, May lee(0503292E)
TG02