Practical 1- PCR and vector PDF

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Date: 29.09.20

Practical 1- Digest plasmid vector & PCR for the insert DNA Title: An experiment to digest the pBluescript plasmid (vector molecule), and to amplify the S-surface glycoprotein DNA fragment of the Covid-19 virus (insert molecule), through the technique of Polymerase Chain Reaction (PCR). Aims: 1) Plasmid vector digest- To cut the pBluescript vector using the restriction enzyme EcoRI, creating an opening in the plasmid, where the viral S-glycoprotein fragment will be inserted and ligated, forming a fusion protein containing viral DNA. 2) PCR- To amplify and clone the viral S-glycoprotein fragment, contained in the pGEM.SGP vector, via the scientific process of PCR. Introduction: The SARS-CoV2 virus is composed of single-stranded, positive-sense RNA. The genome of the Covid-19 virus is extremely large, around 30 kb in size. The virus is comprised of numerous proteins, four of which are structural. These four structural proteins include the membrane protein (M), the envelope protein (E), the nucleocapsid (N), and finally, the spike surface glycoprotein (S). All four proteins play an essential role in the construction and transmission of the virus. It is the S-glycoprotein structure which is ultimately responsible for the infection of healthy host cells with the virus. The spike protein is located on the outside of the virus, where it can directly attach to the host cells, targets; most commonly this is the Angiotensin-Converting Enzyme 2 (ACE2) receptor (Dhama et al., 2020; Li et al., 2020). Once, the virus has invaded the cell, transcription occurs through the replication-transcription complex (RCT), and viral polyproteins are synthesised (Cascella et al., 2020). In conclusion, the structure and function of the S-glycoprotein molecule must be studied due to its potential in the development of future diagnostic tests (antibody tests), and potential cures (vaccines). The overall aim of this experiment is to clone a fragment of DNA which codes for the SARSCoV2 S-glycoprotein. The investigation is comprised of eight smaller practicals, and the first practical is divided into two parts. The first part of practical one is to re-clone the pBluescript plasmid vector from the pGEM.SGP vector. The pGEM.SGP vector already contains the DNA fragment for the spike glycoprotein. However, to successfully amplify and purify the DNA, it must be transferred into a new plasmid (pBluescript) vector. Therefore, the pBluescript vector will be cut open using the restriction enzyme EcoRI. It will create an opening where the viral DNA fragment will be inserted and later ligated. The pBluescript plasmid contains the genetic sequence for a histidine-Taq (His-Tag) within its circular structure. When transcribed, His-Tag results in the sequence of histidine residues (typically six), either at the -N or -C terminus of a protein molecule (Hochuli et al., 1988). Therefore, If the cloning of the vector is successful after the viral DNA has been inserted and ligated into the molecule, the His-Tag and spike protein will be fused, creating a fusion protein. His-Tags are used in to identify and purify fusion proteins. Thus, this method will be used in future experiments to purify the S-glycoprotein DNA from the sample mixture. The second part to practical one is to amplify the S-glycoprotein fragment through PCR. The DNA for the spike protein is already contained within the pGEM-SGP vector (template) molecule. However, to obtain sufficient copies of the S-glycoprotein, it will undergo numerous cycles of replication through PCR. PCR is a quick and easy enzymatic assay that works by replicating specific sequences of DNA (tens of thousands). All PCR assays require

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the following reagents; primers, DNA polymerase (normally Taq polymerase), DNA nucleotides and template DNA. Primers are short sections of DNA that have a specific base sequence that is complementary to the target DNA, i.e. the spike protein (Garibyan and Avashia, 2013). Deciding what primer(s) to use is critical, as it adds additional nucleotide sequences to either end of the recognition sites for the restriction enzyme. It will be these recognition sites that will facilitate the cloning of the PCR glycoprotein DNA, into the pBluescript vector. In both parts of experiment one, the EcoRI restriction enzyme will be used. By using this enzyme, it creates complimentary 'overhangs' or 'sticky ends' in both the pBluescript vector and the spike DNA fragment. Additionally, the primers that will be used in experiment act as EcoRI recognition sites, allowing for the viral DNA to be inserted into the pBluescript vector seamlessly.

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Plasmid vector (pBluescript) digest

In this first experiment, two enzymes (EcoRI & PStI) were considered to be used as the restriction enzyme. Ultimately, it was EcoRI that was chosen for this practical. This is because the PStI enzyme would create a shift in the 'reading frame' when deciphering the genetic code of the S-glycoprotein fragment. Therefore, if PStI enzyme were to be used, it would create a new codon arrangement, and the newly synthesised polypeptide chain would not preserve the six-histidine residues from the original DNA fragment. The PStI enzyme would disrupt the formation of the six corresponding histidine molecules in the polypeptide chain. Maintaining the integrity of the six histidine residues is essential for the later practicals, i.e. purification of the DNA. Therefore, EcoRI is used as it preserves both the 'reading frame' of the glycoprotein and the histidines in frame.

*Draw an image of the EcoRI reading frame, and insert to replace this image.* Materials:        

Test tubes pBluescript plasmid (250 ng/µL) EcoRI enzyme Restriction enzyme buffer Molecular grade H2O Micropipettes Pipette tips Incubator at 37oC

Note: All reagents should be kept on ice, and the restriction enzyme should be returned to the freezer as soon as possible. Methods:

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Digest 2 µg of the vector (pBluescript) plasmid, with the EcoRI restriction enzyme. The final total volume, when all reagents are added, should equal 25 µL.

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Label a test tube, which will contain the plasmid vector digest. Denote with pBS, for the pBluescript enzyme.

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When adding the enzyme, and other reagents to the test tube containing the plasmid, make sure to mix every component in evenly.

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When adding a new substance, use a clean pipette tip and mix the solution by raising and lowering the tip of the pipette into the liquid.

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Therefore, to a clean test tube add:

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8 µL of pBluescript DNA (original plasmid conc, 250 ng/µL) 1 µL of EcoRI restriction enzyme (20 units) 2.5 µL of restriction enzyme buffer 13.5 µL of Molecular grade water to make up the total volume to 25 µL *For calculations please refer to the appendix*

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Incubate the digest at 37oC for approximately 1 hour.

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After one hour, inactivate the restriction enzyme digest by heating the solution 65 oC for 20 minutes.

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PCR to amplify to s-glycoprotein

Materials:           

PCR machine PCR tubes x3 pGEM.SGP plasmid (conc 25 ng/µL) "Forward" primer GAATTC (10 µL) "Reverse" primer CTTAAG (10 µL) Enzyme buffer + Taq polymerase Molecular grade H2O Micropipettes Pipette tips Test tubes Glass beakers

Method:

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Label the three 200 µL PCR tubes with initials.

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Collect three glass beakers to dilute the pGEM.SGP plasmid template.

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Each time the solution is diluted with H2O, use a new pipette tip on the micropipettes.

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Mix the solution by raising and lowering the tip of the pipette into the liquid. Do this for all reagents.

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Pipette 5 µL of the 25 ng/µL pGEM.SGP plasmid into the glass beaker. Add 495 µL of H2O to create a 1:100 dilution.

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Into another beaker, mix 5µL of the 1:100 diluted pGEM.SGP with another 495 µL of H2O to create a 1:10,000 dilution.

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Into the last beaker, mix 5 µL of the 1:10,000 diluted pGEM.SGP with 245 µL of H2O. The final step is a 1:50 dilution. Therefore, the overall dilution is 1:500,000. The final template concentration should be 5x10-5 ng/µL.

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Therefore, when 30 µL of the solution is pipetted from the final diluted solution, it should contain 0.5 pg. of pGEM.SGP plasmid. *For calculations please refer to the appendix*

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Add the 30 µL pGEM.SGP plasmid into a clean beaker. Then add the following reagents:

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3 µL of the "Forward" primer 3 µL of the "Reverse" primer 40 µL of the Enzyme buffer + Taq polymerase 4 µL of Molecular grade water to make up the total volume to 80 µL.

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Using a new pipette tip, pipette 25 µL the reacting mixture into each of the three PCR tubes.

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Then place the three PCR tubes into the PCR machine, and allow the program to run.

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Record on a sheet of paper, the position of the PCR tubes in the machine.

PCR program:    -

First cycle: 3-minutes at 95oC (hot start) 35 cycles: 1-minute 95oC (denature) 1-minute 60 oC (anneal primers) 1-minute 72 oC (extend with Taq polymerase) Final cycle: 10-minutes 72 oC (final extension with Taq) 4 oC “hold”

Results:

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observations: After one hour, the restriction enzyme digest was inactivated by heating the solution to 65oC for 20 minutes. This was to prevent the enzyme from digesting the incorrect region of the DNA fragment and to cut the correct length of the S-glycoprotein fragment. When the enzyme buffer + Taq polymerase solution was added to the master mix (diluted pGEM.SGP, forward & reverse primers, and H2O), the solution turned red.

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Discussion: If both parts of experiment one were successful, then the pBluescript vector has been partially digested, and amplification of the pGEM.SGP vector has led to the cloning of its structure and the viral DNA insert. This will result in a sample with a large source of the Sglycoprotein fragment DNA. In later experimentation, it is this S-glycoprotein fragment that taken will from the pGEM.SGP plasmid and re-inserted and ligated into the pBluescript vector. The future practicals are dependent on the results of practical one.

06.10.20

Practical 2- Vector Dephosphorylation & to 'clean' the Restriction Enzyme digest for PCR product. Title: An experiment to dephosphorylate the pBluescript vector (pBS) using the enzyme phosphatase, and to 'clean' the amplified S-protein DNA for later insertion into the pBS plasmid, creating a recombinant protein. Aims: 1) Plasmid vector digest- To successfully dephosphorylate the pBluescript vector, preventing the cohesive ends of the plasmid from self-ligating, and to form the recombinant protein, which is composed of the pBS vector and viral spike protein DNA. 2) PCR- To digest the PCR product using the restriction enzyme EcoRI, and to purify the viral DNA by removing the remaining enzyme and cleaved protein fragments, using a purification column resulting in a pure sample of S-glycoprotein DNA.

Introduction: For successful cloning of a DNA fragment when inserted into a vector molecule; it is the level of vector preparation that constitutes to the success rate of the cloning itself. When a vector experiences self-ligation or inefficient digestion, typically, there is lower cloning efficiency. In experiment one and two, the pBluescript vectors and the spike protein fragments were both digested using the restriction enzyme, EcoRI. This results in both structures having complementary cohesive ends. Therefore, the vectors should be dephosphorylated to prevent self-ligation from occurring. In the first part of experiment two, the pBluescript plasmids will be dephosphorylated, using the enzyme alkaline phosphatase. The enzyme works by removing the phosphate groups from the five-prime ends (5') of the doublestranded DNA. The plasmid DNA still has the potential to self-ligate. However, with the absence of the phosphate groups at the 5' ends, the strands can no longer covalently bond. This will ultimately leave the strands separated. If dephosphorylation of the molecule did not occur, then ligation of the complementary ends, in the same structure (i.e. pBluescript plasmid), would be more efficient when compared to a vector and an insert (viral DNA) molecule (Optimal dephosphorylation protocols using Anza Alkaline Phosphatase, 2016). Interestingly, if only one of the phosphate groups at the 5' end is removed, then the two strands of DNA can no longer self-ligate. Thus, dephosphorylation of only one 5' nucleotide, will favour the ligation between the vector molecule and the viral DNA insert.

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In the second part of practical two, the PCR products must be digested, to separate the viral DNA fragments from the pGEM.SGP vector molecules. The final PCR product will contain Sglycoprotein DNA fragments, excess restriction enzyme and cleaved protein fragments. It will be in the final stages of experiment two, where the PCR DNA will then go on to be purified. The DNA must be purified before it can be cloned in later experimentation. If the final product is contaminated, it will adversely impact future results. DNA can be purified using a fast and easy technique known as solid-phase Nucleic Acid Extraction. Many companies manufacture commercial use extraction kits, and in this experiment, the 'PCR Purification Kit' from Qiagen will be used. The purification process will involve a silica purification column, QIAquick. Using this column, it will separate the contaminated 'flow-through' solution from the viral DNA. When the PCR sample + PB buffer is eluted onto the surface of the silica column, the viral DNA fragments will bind to the antibodies impregnated into the surface of the column. The DNA will not only be drawn to the antibodies, but also to the high affinity shared between the negatively charged DNA sugar-phosphate backbone, and the positively charged silica particles. The PB buffer acts as a 'binding solution'. It will help the viral DNA attach more readily to the QIAquick column. The 'waste' products, i.e. an enzyme and Protein fragments, will be removed when the PE buffer is applied to the column. The flow-through solution is collected at the bottom of the vessel and then discarded. Finally, the QIAquick column will be rinsed with elution buffer to release the DNA. The final product will contain purified spike protein DNA fragments (Tan and Yiap, 2009).

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Dephosphorylation of the RE-digested pBS plasmid

Materials:       

pBS digest Micropipettes Pipette tips dH2O AP/SAP buffer AP/SAP enzyme Microfuge tubes

Method:

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Make sure NOT to treat the insert PCR DNA, only dephosphorylate the pBluescript vector DNA.

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Collect the sample of pBS digest, and measure the volume of the digested plasmid solution. The total volume should equal 25 µL. However, the actual volume may differ due to evaporation of the sample.

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Therefore, add the appropriate volume of dH2O (molecular water), until the volume is 40 µL (40 µL- 25 µL = 15 µL). This will give a final concentration of 50 ng/µL.

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Remove 28 µL of the digested pBluescript vector DNA and pipette it into a new microfuge tube. Label the tube with pBS ES. This tube should be placed on ice at all times.

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Note that the phosphatase enzyme and buffer may be labelled with AP(alkaline phosphatase) or SAP (shrimp alkaline phosphatase).

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Pipette 4 µL of the AP/SAP buffer- the buffer is at 10x concentration.

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Pipette 4 µL of the AP/SAP enzyme (4 units).

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Pipette 4 µL of molecular grade dH2O to make the total volume up to 40 µL.

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Incubate the solution at 37oC for one hour.

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After one hour has passed, inactivate the enzymes by heating it to 75oC for 20 minutes.

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Save the remaining 12 µL of RE-digested plasmid (pBS.E) as a control.

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Digest of the PCR product

Materials:      

PCR samples x3 RE buffer EcoRI enzyme Micropipettes Pipette tips Incubator at 37oC

Method: 

Combine all three PCR volumes and measure the total volume. The total volume of the PCR digest should be 75 µL, but again may be lower due to evaporation. If higher, it is due to water entering the sample from the water bath.

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Label the tube with pBS E.

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Set up the RE digestion on the PCR product using 1 µL EcoRI enzyme, and 8 µL of the RE buffer.

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Digest the digest at 37oC for one hour.

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Purify the PCR product on a column

Materials:          

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PCR product Buffer PB Buffer PE Micropipettes Pipette tips QIAquick column 2 mL collection tube Centrifuge 1.5 mL microcentrifuge tube x2 Elution buffer

Method:

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Make sure to use clean tips for every new solution and mix the components by raising the pipette tips and down into the solution.

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All centrifugation steps are carried out at 17,900 x g (13,000 rpm). Centrifuge the PCR sample, to mix well.

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Measure the total volume of your PCR product from the digest; this should be around 84 µL in volume. Check using the micropipette at 84 µL.

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Pipette x5 volume of buffer PB to the PCR sample, and mix well. (For example, if you have 100 µL of PCR, then you have 500 µL of the buffer. Therefore, the volume should be around 420 µL.

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Place a QIAquick spin column in a 2 ml collection tube.

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Then using a pipette, add the mixed PCR sample/PB buffer solution to the column. This should be around 504 µL. Put pipette to 600 µL, to collect all of the solutions.

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Place the column into a centrifuge and spin for 60 seconds.

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When placing the sample into the centrifuge, be sure to balance the tube with a second tube. The tube should be of equal weight, and in the same but opposite location in the centrifuge. Make sure to fit the inner lid before turning the machine on.

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Once 60 seconds has passed, discard the flow-through solution (red-solution). A white layer should have formed inside the QIAquick column.

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Place the QIAquick column back into the same 2 mL tube.

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Add 750 µL of buffer PE onto the QIAquick column and centrifuge for 60 seconds.

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Once 60 seconds has passed, discard the flow-through, and the 2 mL collecting tube.

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Place QIAquick column in a clean 1.5 mL microcentrifuge tube, and label the tube with PCR 1E, 41, LG. Make sure to leave the cap off of the tube, place another empty column directly opposite the column containing the liquid, crossing the tube caps.

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Centrifuge again for 60 seconds to remove any excess PE buffer.

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Place the QIAquick column in another clean 1.5 mL microcentrifuge tube. Label tube with PCR 2E, 41, LG.

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Add to the tube 40 µL of elution buffer to the centre of the QIAquick membrane. Then, let the column stand for one minute and then centrifuge the column for 1 minute. Dispose of the microcentrifuge tube.

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Make sure that the elution buffer is poured directly onto the QIAquick membrane, for complete elution of the ...