PCR (Polymerase Chain Reaction) is the quick and easy method of making unlimited copies of any fragment of DNA. Since it’s first introduction ten years ago, PCR has very quickly become an essential tool for “improving human health and human life (TPCR)”. Medical research and clinical medicine are profiting from PCR mainly in two areas: detection of infectious disease organisms, and detection of variations and mutations in genes, especially human genes. Because PCR can amplify unimaginably tiny amounts of DNA, even that from just one cell, “physicians and researchers can examine a single sperm or track down the elusive source of a puzzling infection” (TPCR)”. These PCR- based analyses are proving to be just as reliable as previous methods-sometimes more so and often much faster and cheaper.
Polymerase chain reaction (PCR) is a technique used “to amplify the number of copies a specific region of DNA (Brown)”, in order to produce enough DNA to be adequately tested. This technique can be used to identify with a very high-probability, disease-causing viruses, bacteria, a deceased person, a criminal suspect, and also in the event of an outbreak, “Real-Time PCR can effectively monitor the success of clean-up efforts (RAL,Inc)”.
In order to use PCR, one must already know the exact sequence of a gene or the sequence of interest that lie on both ends of the DNA. While similarity among genes of organisms exists, there will always be genes whose DNA sequences differ from each other. By identifying the genes that are different and unique, one can use this information to identify an organism.
DNA is a double-stranded, “consisting of two such nucleotide chains that wind around each other in the famous shape known as the double helix (TPCR)”. DNA consist of Adenine, Thymine, Cytosine, and Guanine components which can be arrange to generate a “sentence” of a gene sequence which can consists of either a few or thousands of letters long.
To get this copying process started, a template molecule of the DNA or RNA you want to copy is required along with two primer molecules that make up the strands of all genetic materials.
These primer molecules consists of about 20 letters long, which can be linked together in the order desired by a DNA-synthesizer “which add and link one letter at a time (Brown)” to generate the primers needed to start PCR.
There are three major steps in PCR that must be met in order for the process to be successful.
Amplification reaction was done in a 25.0 µL reaction mixture containing 0.4 µL DNA (from DNA extraction), 5.0 µL of 10X PCR reaction buffer, 14.2 µL of sterelized dH2O, 2.0 µL of magnesium chloride (MgCl2, 25 mM), 1.0 µL nucleotide/dNTP mix (10 Mm), and 0.4 µL of 5 u/µL Taq DNA polymerase for each primer namely respectively. The components and the volume used for the amplification reactions are listed in Table 3.2. For the reaction, PCR reaction was performed in a programmable gradient-enabled thermocycler (Bio-Rad MyCycler™ Thermal Cycler).
imported into an aparatus using gel electrophoresis to compare the sample of DNA to other
PCR or polymerase chain reaction is not a DNA typing technique, but a variety of different DNA tests (Riley). PCR duplicates and increases the quantity of a DNA strand which is beneficial to forensic scientists who are faced with little quantity of materials (Saferstein 394). The introduction of PCR-based testing in DNA analysis required scientists to switch to smaller targets that had the same repetitive variation (Jones). This is how short tandem repeat, the newest method of DNA typing,
"Polymerase Chain Reaction (PCR) Fact Sheet." National Human Genome Research Institute. 10 Dec. 2007. National Institutes of Health. .
In order to do this a polymer of DNA “unzips” into its two strands, a coding strand (left strand) and a template strand (right strand). Nucleotides of a molecule known as mRNA (messenger RNA) then temporarily bonds to the template strand and join together in the same way as nucleotides of DNA. Messenger RNA has a similar structure to that of DNA only it is single stranded. Like DNA, mRNA is made up of nucleotides again consisting of a phosphate, a sugar, and an organic nitrogenous base. However, unlike in DNA, the sugar in a nucleotide of mRNA is different (Ribose) and the nitrogenous base Thymine is replaced by a new base found in RNA known as Uracil (U)3b and like Thymine can only bond to its complimentary base Adenine. As a result of how it bonds to the DNA’s template strand, the mRNA strand formed is almost identical to the coding strand of DNA apart from these
limitations” with his invention of the Polymerase Chain Reaction or “PCR” (240). As Silver describes
Illumina sequencing is an example of next-generation sequencing method. It uses fluorescence-based sequence-monitoring technology and contributes to about 90% of current sequencing data2. In Illumina platform, vast numbers of short reads are sequenced in a single stroke. To do this, the input sample must be cleaved into short sections firstly and the length of these sections will depend on the particular Illumina sequencing machine used. In Illumina sequencing, 100-150bp reads are used, somewhat longer fragments are ligated to generic adaptors and annealed to a slide using the adaptors. PCR is then carried out to amplify each read, creating a spot with many copies of the same read. They are then separated into single strands for sequencing3.
Q1Aamp DNA mini kit, leading to the second period being the amplification of 16s rDNA consensus sequence by PCR, particularly using the primers RW01 and DG74. The unknown sample is then taken for gel electrophoresis to confirm and purify the amplified 16s rDNA fragment, done in the third period of the experiment. Once the running of the gel is completed, a cut of the 370bp PCR fragment is taken, and is put for purification of 16s rDNA fragment by the QIAquick gel elution kit, allowing the DNA to elute at the bottom of the microcentrifuge tube
Gel electrophoresis is used in a variety of settings, particularly in molecular biology. Besides being used to separate nucleic acids, such as DNA and RNA, gel electrophoresis is also employed to divide proteins (Gel Electrophoresis). According to research, electrophoresis is applied for the following reasons, "To get a DNA fingerprint for forensic pur...
Lyons, R.H. (2004). How do we Sequence DNA? In A Primer in DNA Structure and Function. Retrieved from http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
In an article concerning Familial DNA Testing, A. Pattock (2011) examines the methods involved in DNA testing. Short Tandem Repeats (STRs), are non-coding genes that have stretches of varying numbers where copies of genetic sequences appear (p.854). STRs are identifiable quantities that can be used as a marker to make the comparison. Each STR forms an allele, which is part of a gene that is formed by a mutation and is found in the same position on a chromosome in every human (p.855). The loci, or fixed position, are the key to using alleles for DNA identification. Although all of the STR alleles put together comprise completely different people, not all of the STRs are unique. Because of this, multiple STR alleles need to be dissected (p.855, 856).
It is used in many labs and only requires the DNA in question, primers that anneal to the beginning and end of the target genes, Thermus aquaticus, Taq DNA, a heat stable DNA polymerase and all four of the deoxyribonucleate triphosphates. There are three steps in the PCR reaction denaturation, hybridization and DNA synthesis. During these steps the DNA is separated or denatured into two strands, hybridized, where the two single strands are complimentary paired to the respective primers, and then the DNA is synthesized with Taq DNA. This is considered one cycle, and it can commonly take 50 cycles to amplify enough DNA to be used. When the PCR is completed a gel electrophoresis is run. The PCR product is put in a specially formed agarose gel that will allow electricity to flow around the gel and DNA and force the DNA to travel down the gel resulting in white bands depending on their electronegativity. When the DNA is transformed from plasmid into the yeast we use salmon sperm to protect the nucleus from becoming degraded and the plasmid lost. This increases the efficiency of the DNA because the sperm DNA will adhere to the yeast cell wall and allow the plasmid to bind to the
The scientific and medical progress of DNA as been emense, from involving the identification of our genes that trigger major diseases or the creation and manufacture of drugs to treat these diseases. DNA has many significant uses to society, health and culture of today. One important area of DNA research is that used for genetic and medical research. Our abi...
RNA cannot serve as a template for PCR, the first step in an RT-PCR assay is the reverse transcription of the RNA template into complimentary DNA and followed by its exponential amplification in a PCR reaction. Usually, this involves the use of dedicated RNA and DNA-dependent DNA polymerases, either in separate (‘two-enzyme/two-tube’) or in single (‘two-enzyme/one-tube’) reactions, as the use of dedicated enzymes with different proper...
DNA is ran through a process called gel electrophoresis in which DNA is cut up and ran through a gel where certain lengths of DNA become banded. This procedure can match a parent to offspring or compare missing persons to skeletons at a crime scene. In this case, “Skeleton A”’s DNA will be matched up to the two missing persons in the St. Charles area. The first step of gel electrophoresis is to create multiple copies of all the DNA (scientifically called Polymerase Chain Reaction) so if something goes wrong, it can be tested again and again. The next step involves digesting the DNA samples with restriction enzymes. In this DNA test, restriction