Title: Simulation of a genetic test for genetic disease screeningIntroduction: Aim of the experiment: Isto perform a simulation of a DNA based test for a genetic disease screening using PCR (Polymerase Chain Reaction), Digestion of PCR products and run the products on a Gel electrophoresis.Genetic testing is a form of a medical test that identifies advancements in chromosomes, genes, or proteins. The results of a genetic test can eliminate or confirm a genetic condition or help determine a person’s chance of developing or passing on a genetic disorder [1].The Fundamental behind the genetic testing is to determine the genetic disease of the patient sample provided, using methods of Polymerase Chain Reaction (PCR) which amplifies a gene or DNA fragment of interest, from DNA, to be cloned. To generate compatible ends, it is common to add restriction sites to the 5’ end of both PCR primers. The DNA is then cut at a distinct point to form smaller fragments using restriction endonucleases, the fragments that are smaller in size are used to conduct gel electrophoresis to separate the fragments by their size and visualise them. The technique used is known as PCR which was used in the experiment, it selects a fragment of DNA and makes multiple copies of the specific region in vitro and uses a reaction mixture which is made up of; DNA polymerase, also known as Taq Polymerase which is isolated from the heat tolerant bacterium Thermus aquaticus. Free nucleotides and DNA primers which are short sequences of nucleotides are 20 in length. Normally two primers are used in each reaction of PCR and bind to the complimentary bases of the template DNA. The reaction mixture is then cycled through temperature changes to design multiple copies of the specific region of DNA being tested. Taq polymerase is ideal for the fluctuation in the temperature, Due how it is heats stable and is most effective at 70 degrees [2]. The fundamental step of PCR is denaturation of the DNA strands to make a single strand template at 96 degrees by breaking the hydrogen bonds apart. The second step is the annealing primers to their complimentary base pairs on the single strand template, to do this the temperature is cooled to 55-65 degree for them to bond. The final step is to raise the temperature to 72 degrees again which is the optimal temperature for the Taq polymerase to extend primers and synthesise new strands of DNA. It goes in a cycle and it is repeated around 25-35 times, it takes around 2-4hours depending on the length of the DNA region that is being copied.  Restriction enzymes are also vital in PCR reaction as it is used to create compatible ends on PCR products. One or more restriction enzymes are used to digest the DNA resulting in either non-directional or directional insertion into the compatible plasmid. Genomic DNA is digested with restriction enzymes that recognize 6-8 consecutive bases, recognition sites occur less frequently in the genome than 4-base sites and result in larger DNA fragments. Preferred insert size for the clone library determines which enzymes are selected, as well as the digestion conditions. Restriction enzymes have a recognition site within the multiple cloning site are used because they do not cut elsewhere in the vector DNA and typically produce two easily resolved DNA fragments. The gene of interest is most commonly subcloned into an expression vector for improved protein expression and or addition of a purification tag. Gel Electrophoresis was also used in the experiment, it is a method used in a laboratory which is used to separate mixtures of DNA, RNA, or proteins according to molecular size. In electrophoresis it involves an electrical field; the field is applied that one end of the gel (the gel being made up of a polysaccharide agarose and buffer) has a positive charge and the other end has a negative charge. Because both DNA and RNA are negatively charged molecules, they are pulled toward the positively charged end of the gel [3]. The molecules that are separated are moved by an electrical field through a gel that contains small pores, molecules move through the pores in the gel at a speed that is associated to their lengths, this means small DNA molecule travels further through the gel than a larger DNA molecule. Bands representing molecules of different sizes can be detected, Bands were observable due to DNA binding loading dye, when a gel is stained with a DNA-binding dye and placed under UV light, the fragments will glow. A distinct “line” of DNA on a gel is called a band. Each band contains numerous amounts DNA fragments of the same size that have all travelled as a group to the same position[4]. Material and method:Method:Part A: PCR amplification of a gene involved in a genetic disease Quantities were calculated using the C1 x V1 =C2 x V2 formula for the master mix.Calculation of Temperature for Thermocycler were also calculated to perform DNA amplificationsCalculations can be seen on the table below:Reagents:Starting Concentration:Final Concentration:Quantity for 1 reaction:Quantity for 5 reactions (MASTER MIX):Red mix(RM)2X1X12.5ul62.5ulForward Primer10uM0.2uM0.5ul2.5ulReverse Primer10uM0.2uM0.5ul2.5ulDistilled water—–—– 1ul—– —–—–10.5ul52.5ulTotal—–—–25ul120ulStep number Step typeTemperatureTime 1Activation941min2Denaturation 9415s3Annealing 6115s4Extension7210s5Go to step 2 for 29 times  5Final extension721min                          Table 1 quantities to prepared to make master mix and  PCR cocktail mix                                    Table 2- The setting for the thermocycler to perform DNA amplifications (Describe accurately the making of the master mix and the PCR profile)Preparation of the PCR reaction:The Final mix was prepared which contained enough component for 5 reactions. 4 PCR tubes were labelled 1-4, the last PCR tube was labelled Master mix (M). Master mix was made by pipetting  62.5ul of Red mix into the tube labelled M, afterwards  2.5ul of forward primer was added also pipetted into (M). Soon after 52.5ul of distilled water was also pipetted into (M). Patient A, B and C were put aside. Tubes with the prepared master mix was vortex held at a slight angle and then centrifuged. Centrifuge was balanced with another aliquot with equal weight for 5 seconds. 24ul of master mix was pipetted to each aliquot labelled 1-4. 1ul of Patient A DNA was pipetted to tube 1, An additional 1ul of patient B DNA was pipetted to tube 2. 1ul of DNA patient C was pipetted into tube 3. Finally, 1ul of distilled water was pipetted into a negative control which contained no DNA this was tube 4. PCR tubes was vortexed to ensure the mixture had been mixed together and then put in the thermocycler to carry out denaturation, annealing, and then binding(amplification) as the DNA sequence is made into multiple copies. Annealing temperature was calculated, the temperature Ta chosen for PCR relies directly on length and composition of the primers the suitable temperature was 61 degrees.Part B: Enzyme digestion of PCR products4 PCR products from Part A was used. 4 new tubes were labelled A, B, C and D. When the PCR tubes were out of the thermocycler, 10ul of Patient A was pipetted into PCR tube 1. 10ul of Patient B was pipette into PCR tube 2.  Another 10ul of Patient C was pipetted into tube 3. Lastly another 10ul pf Negative control was pipetted into last PCR tube. Enzyme was centrifuged, afterwards 1ul of enzyme was added by using a pipette to all new tubes labelled 1-4 including Negative control(N) After the enzyme was added the PCR tubes were vortexed and incubated at 37degrees for 20minutes.Part C: Gel electrophoresis of PCR productsWhile the PCR tubes were incubated, an agarose gel was prepared for samples to be run on the gel. 100ml 1x TBE buffer was added to 400ml of distilled water to dilute the buffer to a total of 500ml. An appropriated amount of agarose was weighed out. This was added to a conical flask with 100 x TBE buffer and was mixed to make the 2% agarose. The solution made was microwaved for one minute to allow the agarose to completely dissolve and appear clear and not grainy. When the solution cooled it formed a gel, 3ul of SYBRsafe was added to the solution.  SYBRsaf was added was to make sure the wells will appear visibly under the UV light. Once the SYBRsafe was added, the solution was poured into the mould and the toothcomb was placed on top to create wells and this could set for 20minutes.Part 2: Gel ElectrophoresisWhen the Gel was set the comb was taken out, and the wells were now visible. The gel was covered with an additional 400ml of the running buffer that was remaining into the chambers on both sides of the gel unit covered 2mm of the gel. So, currents could run through. After 2ul of ladder was put at the corners of the tray, this allowed buffer to function. Then using a pipette, 10ul of each of the samples (8 in total) that was prepared were loaded into each well alongside a DNA ladder for comparison. Once loaded the tank was connected to power supply carefully, the gel was run for 110.2V for 20minutes. Bubbles was shown to demonstrate currents that were running through, Once the timer was up the buffer was poured out and gel was removed and taken to UV light box for observation. An image was picked up displaying the bands of the DNA samples.Results:The result that was obtained under UV light:Lane1234(Negative control)ABCD (Negative control)     Results: The table show the order in which the tubes were pipetted and the result when observed under UV light. Eight lanes are numbered on the gel above. (A lane is a corridor through which DNA passes as it leaves a well.)                   Results: The table also shows the order in which the tubes were pipetted and the result for being under the UV lights, in this case 9 lanes were numbered on the gel above. The 4 lanes contained an enzyme and another 4 lanes had no enzyme present. This can be observed from looking at the picture above.                                                                                                                           1 -DNA ladder                                                                                                                           2 – A PCR 750,100                                                                                                                           3 – A ENZYME -500,250,100                                                                                                                           4 – B PCR -750,100                                                                                                                           5 – B ENZYME – 750,500,250,100                                                                                                                          6 – C PCR – 750,10                                                                                                                          7 -C ENZYME 800,100                                                                                                                         8 – D PCR -100                                                                                                                         9 – D ENZYME -100Explanation:A solution that consisted of 2% agarose gel with 500 ml of 1x TBE buffer was used. It was run on the gel at 110.2 V for 20minutes, from observing the results under the UV light from the experiments there are no fragments, they are DNA bands can be seen but there are no fragments present. Lane 1 consisted of 2ul of master mix and 1ul of patient A DNA sample. Lane 2 also contained 2ul of master mix and 1ul of patient B. Also, Lane 3 was tube 3 that consisted of 2ul of master mix and 1ul of Patient C DNA. However, lane 4 only consisted of 1ul of distilled water and no master mix, which acted like the negative control in the experiment, this was used to balance out experiment Lane 5 was tube A which contained Patient A DNA sample and tube 1, In addition to lane 6 it also contained patient B DNA sample and component of tube 2. So, does lane 7, it has the components of tube 3 and Patient C DNA sample. Lastly lane 8 is a negative control that contains only DNA but no patient sample. In comparison to the backup picture provided fragments are present. Normally once the fragments have been separated, we can examine the gel and see what sizes of bands are found on it.Discussion: (Describe the reason behind the inclusion of each sample of the gel, include headings and citings)           1   2     3    4     5    6   7    8    9                            1 2 3 4 A B C D      Back up picture provided, that fragments are visible                      Results under the UV lights that contain no fragments In comparison to the results obtained from the PCR reaction to the backup picture provided are no fragments present in the results unlike the backup picture. Normally during gel electrophoresis, the DNA is separated using agarose gel electrophoresis, the pre-cast wells are made, and the DNA is loaded into the well in the gel and a current applied. RNA and phosphate backbone of the DNA molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. In the Backup picture, lane 1 only contained EasyLadder™. Which is a molecular weight marker especially designed for fast size determination of linear double-stranded DNA fragments on 0.5% to 3% TAE or TBE agarose gels. In Lane 2 which is sample A contains no enzyme, also lane 3 which is also contains sample A has an enzyme. In addition to lane 4 it is sample B with without an enzyme. As you can see Lane 5 it also contains sample B without enzyme, furthermore in lane 6 t is also sample B with enzyme, this can be visualised in the backup picture. However, Lane 8 is a negative control that has without enzymes and lane 9 is opposite as it has enzymes. When the results from PCR experiment is observed there are no fragments visible, this could be due some reasons. Reasons vary to why they are no fragments seen, this could have resulted due to inaccurate use of pipetting although protocol was followed. Another reason why fragments weren’t visualised could be because of sample being contaminated. Both these reasons could have affected the gel electrophoresis and is the reason why desired results weren’t achieved. However, fragment patterns and molecular genotypes can be identified from using gel electrophoresis. One application of gel electrophoresis in DNA analysis reveals an individual’s genotype at a specific genetic locus. Gel electrophoresis is used to observe these DNA segments and determine if the organism was homozygous or heterozygous with respect to the DNA at that locus. Homozygous individuals have the same DNA sequence at this locus in both chromosomes. Heterozygous individuals have two different versions of this DNA.The molecular genotypes can be seen in the backup picture. Lane 3 and 5 are from individuals that are Heterozygous, and Lane 4 would be from a heterozygous individual.  Lane 4 is a recessive homozygous because there are 2 bands visible, Also the alleles are the same length and they overlap and appear as one in the gel. Lane 3 and 4 are heterozygous because there was more than 1 band produced, as the allele was different lengths In regard to the results from the experiment patient A is a homozygous recessive, Patient B is a heterozygous carrier and Patient C is a homozygous dominant.(Discuss possible diagnosis for patient?)With the use of Gel electrophoresis, it can be used to detect a mutation and diagnose a patient, because a change in DNA sequence will generate a slight change in charge which will affect the migration rate in electrophoresis. Mutations are permanent changes in the DNA sequence in general. This change in nucleotide sequence will change the overall charge of the DNA sequence. This change in charge will affect the migration rate in gel electrophoresis, forming different bands than normal, non-mutant DNA sequence. This is done when DNA fragments are taking to screen for mutations and run in a gel electrophoresis against a normal DNA fragment of the same sequence.A diagnosis the patient could have is SNP (Single nucleotide polymorphisms), which is the most common type of genetic variation among people. Each SNP expresses s a difference in a single DNA building block, called a nucleotide. For example, a SNP may replace the nucleotide cytosine (C) with the nucleotide thymine (T) in a certain stretch of DNA. SNPs occurs commonly throughout a person’s DNA. They occur at least once in every 1,000 nucleotides on average, this means there are roughly 4 to 5 million SNPs in a person’s genome. These variations in the individual may be unique. Henceforth these variations are found in the DNA between genes. There function is to act as biological markers which help scientists locate genes that are associated with disease. When SNPs occur within a gene or in a governing region near a gene, they play a more direct role in disease by affecting the gene’s function. This conclusion was drawn up since PCR products from heterozygous DNA samples exhibit two or more bands , this is seen in lane 3 and 5 in the result of gel electrophoresis under UV light because of the existence of DNA homoduplexes and heteroduplexes.(What is the purpose of using control samples in PCR reactions? And what control sample might have been added in this experiement?)The purpose of controls in PCR is to identify what is needed to amplify. Positive controls are any sample that was amplified, it is useful in this experiment to check that the PCR is worked so that the result of the negative control was negative and was not a failed reaction but negative controls containing no DNA are essential in PCR. It indicates whether there was a proper amplification. When the negative control shows bands of the correct size then you can have confidence that you are amplifying the correct DNA sample or it was contaminated from a previously amplified sample While doing the experiment the negative controls was analysed to verify that no contaminated DNA has been introduced into the master mix or into samples during sample during  processing. A negative control was placed after the last samples.  A positive control was added during the experiment was the samples that contained the enzyme, the positive control consists of a segment of DNA of known size should consist of the same size as the target amplicon. The negative control is only buffers and reagent water. The PCR positive and negative control were used as controls e gel electrophoresis controls.(What was the Ta for each primer and what was the optimal Ta for the provided primers?)The Primer Melting Temperature (Tm) is the temperature at which one-half of the DNA duplex will dissociate and become a single stranded and show duplex stability.To calculate the Ta the melting temperature is needed and is minus from 5. The Wallace rule was used to determine the annealing temperature. Which is Tm=4*(G+C) +2*(A+T). Calculations to determine melting temperature using Forward primer: 5’TGTAAGATGTACACGTTTGCTGC-3’Numbers of A’s r Ts = 13                                                                                  Numbers of G’s or Cs = 10Tm= (4 x no GC bp) = (2 x no AT bp)Tm = (4 x 13) +(2 x 10)Tm= 72Ta =Tm- 5Ta= 72-5Ta=67CCalculations to determine melting temperature using Reverse primer: 5’TCGCTAGATCGAATCGGTAGCTG-3’Numbers of A’s or T’s = 11Number of  G’s or C’s =11Tm = (4 x no GC bp) = (2 x no AT bp)Tm = (4 x 11) +(2 x 11)Tm = 66Ta = Tm-5Ta-= 61CThe optimal Ta in this experiment was 61CThe lowest Tm is used to minus from 5 to get the optimal Ta for the primers.(What are the differences between Taq polymerase and high-fidelity polymerase? And why using Taq polymerase could be a limitation in this test?)Taq polymerase and High-fidelity polymerase are both polymerases that can be used in the experiment both having many differences. Fidelity is fundamental for applications in which the DNA sequence must be correct after amplification. Multiple steps are involved which include having the ability to read a template strand, choose the appropriate nucleoside triphosphate and insert the correct nucleotide at the 3’ primer terminus. By doing this, it effectively rules out the correct and incorrect nucleotide incorporation. This activity is acknowledged as “proofreading,” it is used to excise incorrectly incorporated mononucleotides, which are then replaced with the correct nucleotides. Common examples include cloning/subcloning DNA for protein expression, SNP analysis. High-fidelity PCR utilizes a DNA polymerase with a low error rate and results in a high degree of accuracy in the replication of the DNA of interest. Meanwhile Taq, is a widely characterized polymerase that originates from Thermus aquaticus, lives in hot springs and grows at 70-75(degrees). The Thermophilic bacterium (Taq) consist of a single polpypeptide chain that has a molecular weight of 95kd. Taq has an enzymatic activity at 37°C, although its optimal activity is expressed at a much higher temperature (approximately 72°C). This low-temperature polymerase activity is the fundamental for nonspecific amplification. A thermolabile inhibitor of Taq polymerase is included in the form of a monoclonal antibody, the enzyme does not become active until the inhibitor is heat inactivated. Hence, why Taq polymerase becomes active after the raised temperature destroys the monoclonal antibody during the initial denaturation phase of the PCR reaction which results in release of the functional enzyme. Taq polymarase has an ability to withstand temperatures of up to 97.5°C with an optimum activity range of 75-80°C this makes It ideal for PCR and thus is has become the standard polymerase used in PCR reactions. Taq is commonly because it works well for simple, conventional PCR(What limitation comes with using Taq polymerase in this test?)Unfortunately Taq polymerase has limitations which are the formation of primer dimers, this happens when the activity of Taq peaks at  72°C but is active at lower temperatures, Primers also have a tendency to bind non-specifically at low temperatures, which results in non-specific products in your PCR reaction, and standard Taq only encourages this sort of behaviour For this reason, standard Taq is best used with trusted PCR protocols that don’t need  reams of optimization. A polymerase that is used when need high fidelity is needed is Pfu (Pyrococcus furiosus) DNA Polymerase. It is ideal for individually cloning products for sequencing, and expression experiments. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3? to 5? exonuclease proofreading activity, meaning that it works its way along the DNA from the 5? end to the 3? end and corrects nucleotide-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments have fewer errors than Taq-generated PCR insertsd.The downside of Pfu is its speed which is slower than that of Taq. Combining Pfu and Taq gives you the best option as you get the speed of Taq with the fidelity of Pfu.(What are the differences between ischoziomers and neoschizomers enzymes?)Restriction endonucleases that recognize the same sequence are isoschizomers.  Neoschizomers are a subset of isoschizomers that recognize the same sequence but cleave at different positions from the prototype. Also AatII (recognition sequence: GACGT?C) and ZraI (recognition sequence: GAC?GTC) are neoschizomers of one another, While MspI (recognition sequence: C?CGG) and  HpaII (recognition sequence: C?CGG) are isoschizomers. Isoschizomers are pairs of restriction enzymes that are specific to the same recognition sequence. An example of an isoschizomers that is specific to each other are, Bbu I (CGTAC/G) and Sph I (CGTAC/G) . The first enzyme function is to recognize and cut a given sequence. Isoschizomers are detached -/from different strains of bacteria and therefore require different reaction conditions.Meanwhile neoschizomer is an enzyme that recognizes the same sequence but cuts it differently. Neoschizomers are a specific type (subset) of Isoschizomers. An example of a neoschizomers of each other are Sma I (CCC/GGG) and Xma I (C/CCGGG) .(What are other techniques relative to PCR?)Although the PCR experiment is the easiest to perform it has its advantages and disadvantages. A disadvantage is that the PCR efficiency cannot be 100% reliable due to factors such as the presence of PCR inhibitors or enhancers, RNA extraction, and different uses of probes, primers, and enzymes. These factors also contribute to variations in efficiency PCR efficiencies vary from 60% to 110%. An advantage is that PCR only amplifies specific targets. For the experiment to work primers were used, such primers allow the amplification of nucleic acids of multiple related infectious agents. Also, PCR amplifies only a very limited part of the genome, PCR consists of sets pf multiple primers within a single PCR mixture that produce amplicons of varying sizes that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test-run that otherwise would require several times the reagents and more time to perform. Annealing temperatures are fundamental so each of the primer sets are optimized to work correctly within a single reaction, and amplicon sizes. So that their base pair length is different enough to form distinct bands when visualized by gel electrophoresis. There are Alternative Methods of Polymerase Chain Reaction such as LAMP, NABSA strand displacement and Ligase chain reactionLoop Mediated Isothermal Amplification (LAMP) is a specific, simple, rapid and cost-effective isothermal nucleic acid amplification method. It depends on the auto-cycling strand displacement deoxyribonucleic acid (DNA) synthesis which is carried out for 45-60 min at 60-65°C in the presence of Bacillus stearothermophylus (Bst) DNA polymerase, deoxyribonucleotide triphosphate (dNTPs), specific primers and the target DNA template. The method of LAMP is that a DNA polymerase with high strand displacement activity and a set of four specially constructed primers) that recognize six distinct sequences on the target DNA. The mechanism of the LAMP amplification reaction has three steps, which are the Production of starting material, cycling amplification and elongation and recycling. LAMP is more resistant to compounds present in clinical samples than PCR. LAMP is as suitable for DNA sequencing as PCR. Another method relative to PCR is Nucleic acid sequence Based Amplification, it is designed for the detection of RA targets, DNA can also be amplified.qReferences:[2]Name of organisation responsible for site: KHANS ACADEMYTitle: Polymerase chain reaction (PCR)[ONLINE]Date site was published:2019Date Of viewing site:  1/2/2019URL: https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr Research gate Title: Controls of PCRDate site was published: 2019Date of viewing site: 1/2/2019URL:https://www.researchgate.net/post/what_are_controls_in_PCR_if_we_want_to_get_positive_control_then_how_can_we_get_i_mean_comercially_available_or_from_where_we_gotScience DirectTitle: Genetic screeningDate site was published: 2019Date of viewing site: 1/2/2019Url: https://www.sciencedirect.com/topics/medicine-and-dentistry/genetic-screeningNew England biolabsTitle: Restriction enzymesDate site was published:2019Date of viewing site:2/2/2019URL: https://international.neb.com/applications/cloning-and-synthetic-biology/dnaBlog SinaTitle: Isoschizomer, neoschizomer & isocaudomerDate site was published: 2019Date of viewing :2/2/2019Url: http://blog.sina.com.cn/s/blog_5fe22e470100hm78.htmlGenetic Home referenceTitle: single nucleotide polymorphisms SNPSDate site was published: January 29, 2019Date of viewing: 2/2/2019URL: https://ghr.nlm.nih.gov/primer/genomicresearch/snphttps://www.sciencedirect.com/topics/medicine-and-dentistry/genetic-screeninghttps://international.neb.com/applications/cloning-and-synthetic-biology/dna-preparation/restriction-enzyme-digestionhttp://blog.sina.com.cn/s/blog_5fe22e470100hm78.htmlhttps://ghr.nlm.nih.gov/primer/genomicresearch/snp.https://www.sciencedirect.com/topics/neuroscience/taq-polymerasehttps://www.zora.uzh.ch/id/eprint/116536/1/Favrot_2015_Buenos_Aires_Polymerase.pdfhttp://flax.nzdl.org/greenstone3/flax;jsessionid=874EBF5E5FFCD575B271A5254A95CD87?a=d&c=BAWEPS&d=D118&dt=simple&p.a=b&p.s=ClassifierBrowsehttp://passel.unl.edu/pages/informationmodule.php?idinformationmodule=1065724861&topicorder=12&maxto=15refrence dr t sagmma lectures  fpr annealing temphttps://socratic.org/questions/how-can-gel-electrophoresis-be-used-to-detect-mutations