PCR: Amplification of blood extracted DNA.
Abstract
To detect the presence or absence of Interferon gamma mutation on +874 it was necessary to run a PCR in order to amplify the DNA that was extracted from whole blood. The PCR products were then run on 1.5% agarose gel so as to clearly visualise the trends in mutation of interferon gamma. Even though it was established that possible allele combination are heterozygous AT or TT/AA homozygous allele. The population on which this lab was done most showed heterogenicity within their DNA as denoted by having visible band on all the 2 well conducted per sample in which each contained a different primer. In this practical lab no individuals were found to have alleles TT and all the other heterozygous for the mutation (Dabora,. et al. 2002).
Introduction
Interferon-gamma (IFNγ) is an important immunomodulatory cytokine produced by activated T cells and NK cells that plays a pivotal role in promoting host defence, it is a dimerized soluble cytokine that is the only member of the type II class of interferons. IFNγ is distinguished from IFNα and IFNβ by its ability to regulate a number of immune functions. IFNγ induces its biologic effects by interacting with a specific IFNγ receptor expressed at the cell surface. IFNγ also called type II interferon, is a cytokine that is critical for inborn and adaptive immunity against viral, some bacterial and protozoal infections. It is an important activator of macrophages and inducer of Class II major histocompatibility complex (MHC) molecule expression. Deviant IFNγ expression is associated with a number of autoinflammatory and autoimmune diseases (Schreiber and Farrar 1993).
Polymerase chain reaction (PCR) is a technique used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The PCR is used to amplify a precise fragment of DNA from a complex mixture of starting material, referred to as the template DNA (Blake., et at.1992).
The amplification of DNA occurs in the cycling phase of PCR, which consists of three stages: denaturation, annealing and extension. Each molecule of DNA is a double helix formed from two complementary strands of nucleotides held together by hydrogen bonds between G-C and A-T base pairs. In the denaturation stage the sample is heated to 94◦C causing the double stranded DNA molecule to melt forming two single stranded molecules. DNA melts at this temperature because the hydrogen bonds that hold the two strands of the DNA molecule together are relatively weak. The temperature is then lowered to a range between 50 – 65OC and oligonucleotide primes anneal to the template. The primers are in molar excess to the template strands and bind to the complementary sequences before the template DNA re-associates to form double stranded DNA. After the annealing of primers, the temperature is then raised to 72OC. Nucleotides are then added into the sample. The enzyme Taq polymerase catalyses the addition of nucleotides to the 3’ ends of the primers using the original DNA strand as a template. This complete a single cycle for the PCR. The thermos cycler is programmed such that the researcher can input the number of cycles they want to be done on their sample.in this case the samples were exposed to __ cycles (Mullis and Faloona 1987).
Aims and objectives
To check for single nucleotide polymorphism (SNP) mutation +874 (positive)
To identify the alleles available in each sample for the mutations.
To determine the genotype which is in each sample that was loaded.
To identify the individuals within the collected sample who have the +874 mutation
To run an electrophoresis gel to determine the positions of the bands in order to deduce the genotype.
To amplify DNA using the PCR method.
Materials and methods
Refer to the practical schedule: Forensic Genetics 2 (BSFS 204) 2016, Practical schedule: Protocol: Practical 2.
Alterations made- 3µl of the ladder was loaded on to the gel for electrophoresis.
10µl of loading dye was mixed with 20µl of the sample and of the final volume 15µl was used to load on the gel.
1.5% agarose gel was used.
The electrophoresis machine was set to 100 volts; 300 amps for 45 minutes.
Results
The following table are a tabulated representation of how the sample we loaded on the gel. The first sample set follows below.
Table of results only available in downloadable document. Search for it here
Wells labelled 0 and 00 both contained a 100 bp ladder. Lane 4; 5;8 and 9 of the first set had smears. While well 1; 3; 10; 11; 12; 13 also had smears for the second set of samples.
Images also available in the downloadable file
Discussion
The PCR follows DNA replication that occurs in cells, however, it does this at a faster rate and this coping of DNA is don’t outside the cell in vitro to amplify exact portions of the DNA strand. Components of the PCR include the template DNA, primers, Taq polymerase, magnesium Chloride, dNTPs and the buffer. All these were added in a single mixture that we termed the master mix.
Following the lab practical that had been conducted last time, the product of the extracted DNA had been stored in the freezer to prevent DNA degradation. This sample of extracted DNA from whole blood was used as the template DNA which is an essential component of the PCR process.
The most important components of the PCR reaction were the two primers, which are short DNA sequences that lead the region to be copied. A primer acts to identify the portion of the DNA template to be copied and short synthetic pieces of DNA that anneal to the template molecule either side of the target region. It is a chemically synthesized oligonucleotide that is added in a high concentration relative to the DNA template to drive the PCR reaction. In this practical the first primer that was used tested for the mutation of the A allele hence it amplifies T allele. And the second primer detected the T mutation hence multiplied the A allele. Both primers were added in equal amounts to the tube. The temperature at which primers anneal to the template DNA depends upon their length and sequence. A basic rule of thumb is used when designing primers to estimate the melting temperature, for each A or T in the primer 2◦C is added to the melting temperature and for each C or G 4◦Cis added, this is because G – C have three hydrogen bonds which are stronger than the two hydrogen bonds on A—T. (Goodwin, et al. 2007)
As for the template DNA that was used, the amount of DNA added to the PCR highly depends on the sensitivity of the reaction. In most forensic cases the PCR is highly optimized so that it can work with low levels of template. “Most commercial kits require between 0.5 and 2.5 ng of extracted DNA for optimum results. This represents between 166 and 833 copies of the haploid human genome – one copy of the human genome contains approximately 3 pg of DNA.” Goodwin (2007). Most forensic profiling can be carried out successfully with fewer templates – even below 100 pg or 33 copies of the genome; however, the interpretation of profiles can become more complex as the amount of template DNA is reduced. However, in this case the template DNA was derived from DNA extracted from whole blood whose extraction had been done in the previous practical lab. Hence the template DNA was not in small quantities as in most forensic cases (Butler, 2007).
DNA replication in cells use the enzyme DNA polymerase and this is the same enzyme that was used in the first PCR experiments. Even though it worked in it had to be continuously added to the mixture because it kept losing its ability to carry out what was assigned to do. This DNA polymerase that was isolated from E. coli, in each cycle of the PCR the enzyme was inactivated by the high temperatures in the denaturation phase and fresh enzyme had to be added. To overcome this problem of enzyme denaturation the use of Taq polymerase was employed. Taq polymerase is an enzyme that was isolated from a thermophilic bacterium, Thermus aquaticus. The Taq polymerase enzyme can tolerate the high temperatures that are involved in the PCR and works optimally at 72–80◦C hence is it ‘heat resistant’. Taq polymerase enzyme exhibits significant activity at room temperature such that it can create non-specific PCR products, adding the enzyme to a pre-heated hot start reaction reduces the non-specific binding and again improves the specificity and yield of a PCR. However other efficient but expensive enzymes have been synthesised like AmpliTaq Gold (Butler, 2007).
The magnesium Chloride was used for stabilizing prime template DNA interaction and for the successful use of Taq polymerase. Without it Taq polymerase would not work.
Interferon gamma polymorphism exists within a population in which different alleles are found within a population. For the alleles TT which is homozygous the IFNy production is increased therefore cytokine release is more efficient thus there is faster response to stimuli caused by the IFNy. This is the case with no individual in the selected population had these pair of alleles. As for the AT and AA genotypes it means that there is low production of IFN and hence immune system is slower in responding to stimuli. Wells labelled 0 and 00 both contained a 100 bp ladder. Lane 4; 5;8 and 9 of the first set had smears. While well 1; 3; 10; 11; 12; 13 also had smears for the second set of samples. Smearing could have been a result of poor loading techniques and volumes.
References
Blake, E., et al. (1992) Polymerase chain-reaction (PCR) amplification and human-leukocyte antigen (HLA)-DQ- ´α oligonucleotide typing on biological evidence samples – casework experience. Journal of Forensic Sciences 37, 700–726.
Butler, J. M. (2007). Fundamentals of forensic DNA typing. Amsterdam: Academic Press/Elsevier.
Dabora, S., Roberts, P. et al. (2002). Association between a High-Expressing Interferon-γ Allele and a Lower Frequency of Kidney Angiomyolipomas in TSC2 Patients. The American Journal Of Human Genetics, 71(4), 750-758. doi:10.1086/342718.
Goodwin, W., et al. (2007) An introduction to forensic genetics. John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA.
Mullis, K.B., and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase catalyzed chain-reaction. Methods in Enzymology.
Schreiber, R. & Farrar, M. (1993). The biology and biochemistry of interferon-gamma and its receptor. Gastroenterologia Japonica, 28(4), 88-94. Retrieved from http://link.springer.com/article/10.1007/BF02782897.
