Using PCR and Gel Electrophoresis to Determine Genotype In certain situations, it is necessary to identify DNA retreived from a sample. When there is a small sample in need of identification, Polymerase Chain Reactions are used to multiply the DNA in the sample in to many identical samples. The DNA retrieved from the reaction can then be imported into an aparatus using gel electrophoresis to compare the sample of DNA to other samples. In our experiment we learned the how to replicate tiny samples of DNA into usable amounts and how to analyze the specimine using gel electrophoresis. The samples of DNA were obtained by plucking individual hairs from students' heads and using the PCR device to replicate the DNA from the roots of the hair. The replicated DNA samples were then placed into the electrophoresis gel and the device was turned on. Using the methods discussed above we found that three of the fourteen samples, 21%, were homozygous and the remaining eleven samples, 79%, were heterozygous. We concluded that it is possible to examine small amounts of DNA by first replicating the sample using Polymerase Chain Reactions then using gel electrophoresis to determine the genotype of the DNA. The main goal for our experiment was to learn how to examine DNA when there is only a small sample present. We examined the samples of DNA obtained from student hair using the replication method of Polymerase Chain Reaction and the inspection method of gel electrophoresis. The way the PCR method works is by first mixing a solution containing the DNA, DNA polymerase primers, and certain nucleotides. Next, the solution is heated to allow the strands to seperate, then cooled to allow the primers in the solution to b... ... middle of paper ... ...q DNA polymerase to each tube while disallowing the tubes to cool and without taking time to mix the reaction solution after adding the Taq polymerase. When the PCR technique is completed, the tubes are stored at 4°C until analysis of the tubes. To alylize the PCR results with the gel electrophorese, 2.5ul of the 10x loading dye is added to each PCR reaction tube. The gel for the electrophorese consists of 1.5% agarose gel with 0.5x TBE and 200ng/ml ethidium bromide. The gels were run at 90-100 volts for 1-1.5 hours. Upon completion of the experiment we were able to examine the DNA. First, the electrophorese revealed that three of the fourteen samples were were homozygous while the other eleven were heterozygous. This conforms the fact that close to eighty percent of our population is heterozygous and twenty percent homozygous (Isprilus, 2000).
The repeat segments are cut out of the DNA strand by a restrictive enzyme that acts like scissors and the resulting fragments are sorted out by electrophoresis (Saferstein 391). However, there are some drawbacks using the RFLP method in the forensic science community. The RFLP technique requires a large amount of DNA and must be of high quality and cannot be degraded (Jones). Forensic scientists and the law enforcement community determined a need for a DNA profiling method that could be used on smaller DNA samples. Thus, the RFLP technique has been almost entirely replaced by Polymerase chain reaction.
We first started out with DNA extraction. We swabbed a toothpick on the inside of our cheek. We isolated the mitochondrial DNA from that sample of cheek cells. We used different reagents that lysed the cells and made it easier for the students to be able to have access to the nucleic acids. For example the buffer ATL breaks down the cell membrane, but has the environment similar to the cell as far as salt and pH concentration. The proteinase K breaks do...
Then, using a fresh tip each sample, I transferred of the enzyme to each separate tube of the DNA samples. By adding the enzymes, this will cut the DNA molecules into small pieces when we place it into the gel and let it
Materials that were used during this “Hair Evidence Lab” included: a microscope, hand lens, tweezers, white paper, cover slips, and sliders. I took a tweezer to pull out a strand of my hair. I tried to pull out one that had a root on it. I then put my strand of hair on the white paper. I had to use white paper because my hair is black and dark. After putting my strand of hair on the white paper, I then put it on a slider. After that, I put the coverslip on top. I then proceded by looking at it on a microscope. I was given a “Hair Evidence Lab” paper, before even putting the microscope on any power. I had to look through the microscope to see what the root of my hair looked like.
... tested hairs and other parts for DNA and concluded that they fit into our family tree. “ Those hair samples that could not be identified as known animal or human were subsequently screened using DNA testing, beginning with sequencing of mitochondrial DNA followed by sequencing nuclear DNA to determine where these individuals fit in the tree of life” (Ketchum 2013).
All individuals, except identical twins, have unique DNA. DNA fingerprinting is an unambiguous identification method that takes advantage of the difference in the DNA sequence. This process of DNA fingerprinting starts with the isolation of the DNA from the identified sample, such as blood, saliva, semen or other body tissues. In instances where the available sample is small for the process of fingerprinting, then the sample is augmented through a process called polymerase chain reaction (PCR). PCR is the process of DNA replication that does not use living cells and, therefore, the process is appropriate in fingerprinting old samples. The focus of this process is on the short tandem repeats (STR) that have short units of DNA that are repeated several times in a row. After the DNA is isolated and amplified it is treated with restriction enzymes. This process cuts the DNA strands at definite sequences called restriction fragment length polymorphisms (RFLPs). Since everybody’s DNA is different the resulting RFLPs will be of different sizes. These fragments are observed in gel electrophoresis; a process that separates DNA based on the size of fragments. RFLP analysis is based on the fact that since everyone’s genetic sequence or the variable number of tandem repeats is different this result in the different sizes of RFLPs. Gel electrophoresis involve the separation of the fragments of DNA as they migrate through an agarose gel when an electric current is applied to the gel. The DNA that has separated is then drawn out of the gel with a nylon membrane which is treated to break the hydrogen bonds holding the DNA strands together. The separated strands of the DNA is then cross-linked to the nylon membrane a...
"Polymerase Chain Reaction (PCR) Fact Sheet." National Human Genome Research Institute. 10 Dec. 2007. National Institutes of Health. .
it can be completed in a few days and it only requires a small amount of DNA,
Ballantyne, Jack, George Sensabaugh, and Jan Witkowski. DNA Technology and Forensic Science. New York: Cold Spring Harbor Laboratory Press, 1989.
Another way to extract DNA is by solution based methods. There are also different ways of solution base methods. The salting out method was used back around 1979. They used glass powder stuff to purify DNA. Similar steps are used through most of the solution extracts so they can contribute the DNA cells evenly. They usually combine nuclear acid. These solutions also use buffering with pacific Ph. to make a chemical change. After many steps through columns and buffering the DNA is later placed in distilled water after it has been cleaned.
Genetic tests use techniques to examine genes or markers near the genes. Direct testing for diseases such as Cystic Fibrosis and Sickle Cell Anemia come from an analysis of an individual's specific genes. A technique called linkage analysis, or indirect testing, is used when the gene cannot be directly identified but can be located within a specific region of a chromosome. This testing requires additional DNA from an affected family member for comparison. Because each person's DNA is unique (except for identical twins), genetic tests also can be used for individual identification ("DNA fingerprinting"). This technique is often used in forensic work, where samples from a crime scene such as blood or semen could be used as incriminating or acquitting evidence.
View gel under UV transilluminator, record data and take photo of gel for further analysis of the DNA bands.
Create wells: put a comb template in the middle of the tray; wait until the mixture becomes solid. After, remove the comb standing straight. 4. Remove rubber ends: transfer the gel tray into the horizontal electrophoresis and fill it with the concentrated electrophoresis buffer. 5. Materials and methods: Experiment: 1st, prepared milk samples should be already done by the teacher.
DNA chips contain a large number (up to one million) of different single-stranded DNA fragments that analysis the genetic information of a cell. These biochips are halted on the substrate surface in discrete spots. To increase sensitivity each spot contains several millions of identical oligonucleotides. The sample which needs to be tested usually labeled with a fluorescent dye and it contains single-stranded genetic chains (DNA fragments, mRNA, or cDNA). The genetic chains become blind to the spot on the substrate when they match with the immobilized oligonucleotides. The biochip is then illuminated with a suitable wavelength, so that the fluorescence light pattern of the different spots allows the determination of the type and concentration of target genetic chains in the sample [2].
...ary part in genotypes of potential interest that human geneticists breeders, as well as evolutionary geneticists are investigating. However, although we have the capability to unravel experiments that the founders of quantitative genetics would have never imagined, but their basic, un-computational machinery that they developed is most easily adaptable to the latest analyses that will be needed. We are far from ‘letting-go’ molecular biologists from the mathematical techniques/systems, because this age in respect to genomics has been forced into accepting gratitude due to the major importance of quantitative methods as opposed to the new molecular genetics. As geneticists tend to map molecular variation as well as genomic data, quantitative genetics will be moving to the front position because of its relevance in this age of rapid advancement in molecular genetics.