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Glutamate is one of the major neurotransmitter in the human central nervous system, which have primarily excitatory synaptic activity. There are 2 broad classes of glutamate receptor, NMDA (N-Methyl-D-Aspartate) and non-NMDA receptors identified from their sensitivity to glutamate. The non-NMDA receptors include Kainate and AMPA (Î±-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid). Both the NMDA and the Non-NMDA channels are permeable to cations but the AMPA receptor channel gates the sodium and potassium flux. The NMDA receptor on the other hand, is normally blocked by voltage dependent Magnesium ions which clogs the channels preventing depolarization by Calcium ions (Nicholls,J et al., 1992).
The NMDA receptor have been identified to have 3 subunits NR1, NR2A-D and NR3. Lipsky et. Al (2003) elaborates that NR1 and NR3 differs from the other NMDA receptor subunit (NR2B) because glycine is the primary ligand for the former whereas the NR2 have the glutamate binding property.
The study of the NR2B subunit of the NMDA receptor have generated a huge interest because of its glutamate binding property which is involve in the long term potentation (LTP) which plays an important role in learning and memory. The gene coding of this particular subunit is GRIN2B. A study conducted by Schito et al (1997) found out that this particular gene is expressed in fronto-parietal-temporal cortex and hippocampus pyramidal cell and at lower levels in the basal ganglia. The Hippocampus is part of the limbic system which derives its name from the curving shape which reminded early neuroanatomist of a rams horn - Ammon's horn. It is subdivided into 2 cell types: Pyrimidal Cells of Ammons Horns which comprises of the Cornu Ammonis fields CA1-CA4 and the Granule Cells of Dendrate Gyrus(DG) - CA4 is frequently associated as part of the Dendrate Gyrus see Fig. 1 (Swan-Wallis, 2010).The gene is located at the 12p12 and 419kb in size which have 13 exonic sequences (Li, D. et al., 2007).
Fig.1 Hippocampal Anatomy (Source: Hesselink, n.d )
Fig.2 Three- Dimensional Modelling of the Hippocampal Subfields.Color-coded 3D models of major hippocampal subfields CA1(red), CA2(dark blue),CA3(green) and DG(yellow). 3D orientation bars:lateral,red;Ventral,green;rostal,blue.Scale Bar,1mm.(Thompson et al., 2008)
Chapter 2: Materials and Methods
These are the materials used for the Polymerase Chain Reaction(PCR) Analysis of Human Gene - GRIN2B for amplification.
Enzyme mix, which contains:
Taq DNA polymerase
Sterile Deionized water.
Oligonucleotide primers (Reverse and Forward primers)
Human placental DNA 1µg/ml
Agarose (1%w/v agarose gel soln.)
Step ladder (blue/red) - 50bp
TBE (Tris-HCL,Boric Acid and EDTA)
Pipettes and tips
Casting tray with comb
2.B.1 Biotechnology Information
Before the actual laboratory work for the analysis of a gene, a gene of interest is researched and selected from established and reliable informatics databases online such as the NCBI (National Center for Biotechnology Information),New Scientists, Nature, Science or even from the media. The gene of choice was research thoroughly from online journals to know region of the gene to amplify that is hypothetically studied so far to be the cause of a disorder.The website OMIM(On-line Medelian in Man) is an excellent website to start getting to know more about the gene make up and location and most importantly the right name of the gene that is need later for downloading the genomic sequence. The Human Genome Project (HGP) website, which is a joint effort by the US Department of energy and the National Institute of health ( http://www.ornl.gov/sci/techresources/Human_Genome/project/about.shtml. Accessed:12 July 2010) is also a reliable source that provides great wealth of information and history as well as links to different bioinformatics site that is of great help throughout this project.
When all the information of the GRIN2B gene has been gathered from the said websites, the UCSC (University of California-Santa Cruz) Genome Bioinformatics gene sorter link will be able to identify the genomic sequence. The sequence was adjusted to up to 50 upstream and downstream base pairs for specificity and efficiency (Igo, RP et al.,2002).Having done the research on what region of the gene that carries the biological role in the disorder selected,the "Primer 3" website - http://frodo.wi.mit.edu/primer3/ - will help design primers for in silico PCR.The product size range was adjusted to 501-100bp amplicons for specificity. In this project, apart from the exon 2 that was amplified it is decided to include a part of the introns sequence so the designed coverage of the forward and reverse primers will include all parts of the exonic sequence that might carry the mutation of the disorder, in this case Schizoprehnia.The "Primer 3" output will yield a forward and reverse primer as well as the annealing temperature, which is vital for the purity and yield of the product (Rhylik,W et al.,1990).
Lastly, with the designed primers an in silico PCR is performed through the UCSC website on the "PCR" link.This yields sequence for the primers as well as its annealing temperatures.The calculations as done as quoted "The temperature calculations are done assuming 50 mM salt and 50 nM annealing oligo concentration. The code to calculate the melting temp comes from Primer3"(Kent, WJ. et al, 2002). Now that the in silico PCR is performed, primers are outsourced from Sigma-Aldrich, a life science and high technology company.
2.B.2 Polymerase Chain Reaction(PCR) Analysis
The aim of this project is to be able to amplify the region of the gene that is the carrier of the disorder and to identify any possible mutations. The Polymerase Chain Reaction (PCR) method is used to amplify small amount of DNA. Firsty, the region of the DNA to be amplified is denatured,wherein the double standed DNA are melted to yield two single strands of complementary DNA called primers that will anneal to the target DNA, then the Taq polymerase will attach to the double stranded template and copies strand(Gardner A. et al., 2000).
2.B.3 PCR Set-Up and Experimental work
Through out laboratory work, Good Laboratory Practice (GLP) is observed to adhere to the standard as practiced in the industry. Lab coats and googles must be worn inside the working area as well as gloves when handling any laboratory equipments and reagents. It must be remembered that in this project all samples should be left in a frozen tray to avoid the degradation of the samples and always fresh pipettes everytime to avoid contamination as it can spoil the whole project and would not yield desired results.
There are two sets of oligonucleotides (forward and reverse) ordered from Sigma-Aldrich with the quality assurance document that gives precise volume to give 100µM concentration. To make the PCR mixture, the solution must be further diluted to 10µM as a working stock solution, to do this in each individual eppendorf tube 10µl of the primers and 90µl of sterile deionized water to make a 10µM working stock solution used in the PCR mixtures. Table 1 details the components to make the master mix, in total only 60 µl is needed but an allowance of 7 µl is considered for pippeting errors.
Forward Primer (10µM) = 1 µl
Reverse Primer (10 µM) = 1 µl
Enzyme Mix (Red) = 10 µl
Sterile deionized Water = 7 µl
Table 1: PCR master mix.
The master mix is further divided into three reaction tube to run the PCR, one of which is the control tube with the sterile deionized water and the remaining two experimental tubes with 1µl of DNA in each of them.
1st Reaction Tube: 20µl http://www.freeclipartnow.com/d/38329-2/eppendorf-open.jpg
19 ul of master mix + 1 µl of DNAhttp://www.biotroninc.com/eng/html/p_img/eppendorf_tube_s.gif
2nd Reaction Tube:20µl
19ul of master mix + 1µl of DNAhttp://www.biotroninc.com/eng/html/p_img/eppendorf_tube_s.gif
3rd Reaction (control) Tube: 20 µl
19ul of master mix + 1µl of Deionized H2O
67 µl total volume of master mix
Fig. Illustration on how the PCR mix are divided into each individual reaction tubes. Human placental DNA have 500ng/ml concentration.
The thermocyler is programmed accordingly as shown in table 2.This program is used to all the PCR runs in the experiment with the different annealing temperatures tested. The PCR runs at approximately 1hr and 39mins to 1hr and 48mins depending on the annealing temperature.
94°C 5 minutes - 1 cycle
94°C 1 minute } 30 cycles
X °C 30 seconds}30 cycles
72°C 1 minute } 30 cycles
72°C 5 minutes- 1 cycle
Table 2: Thermocycler programme for GRIN2B PCR.
2.B.4 Gel Electrophoresis
Gel Electrophoresis provides the location of the DNA fragments. The DNA is negatively charge and by introduction of electrical current the ions migrate to the positive anode of the gel tank (Gardner A. et al., 2000). In this procedure, a 1%w/v agarose gel is prepared (1 g) dissolved in TBE (Tris-HCL,Boric Acid and EDTA) 100ml in a conical flask.The mixture is then slow heated in a microwave oven with constant stirring without allowing it to simmer for the agarose powder to dissolve, this is done at approximately 2mins maximum.The solution is left to cool and adding 10 ul of GelRed, a non toxic dye that would give fluorescence for visualization in the UV light later on.Prepare the casting tray with comb (at least 16 wells for sample loading) for the agarose solution to settle.When the conical is safe to touch pour the lukewarm agarose solution to the casting tray to settle leave it for a at least 30minutes to cool and hardened.By this time the PCR tubes will be ready to take out.Make sure that frozen tray should be used at all times. After taking out the comb from the gel, pour considerable amount of TBE enough to soak the gel a few milimiters from its surface.
Moreover, step ladder (50bp) should be loaded into the first well.Then from the samples take 10ul of each loaded into each of the wells.This procedure should be done carefully avoiding spillage as these can affect the outcome during UV visualization.After loading connect the gel into the electric conductor making sure that sample are on the cathode side (red strip in the casting tray) to allow it to travel to the anode side of the tray at a constant 70V. This will run for approximately 1hr and 30mins.
Chapter 6: Citations
Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. The human genome browser at UCSC. Genome Res. 2002 Jun;12(6):996-1006.
Human Genome Project (2008). About the Human Genome Project. Available at: http://www.ornl.gov/sci/techresources/Human_Genome/project/about.shtml(Accessed: 12th July 2010).
Hesselink, J.R (n.d). The Temporal Lobe and Limbic System.[image online].available at: http://spinwarp.ucsd.edu/NeuroWeb/Text/br-800epi.htm (Accessed: 16th August 2010).
Chapter Seven: Appendices
7.1 Genomic sequence
The genomic sequence of the human GRIN2B Gene with 14 Exons.The amplified Exon 2 are highlighted in red color. The Exons are in upper case and the intronic sequence are showed in lower case. (http://genome.ucsc.edu, Accessed: 14th January 2010)