There are several regions of VNTR's in the genome with repeat lengths ranging from 10 to 100bp. The core repeats are represented in some alleles thousands of times; the variation in repeat number creates alleles that range in size from 500 base pairs to over 30,000 base pairs (The Council for Responsible Genetics). The number of sequences vary quite greatly person to person and the sequences are inherited from your parents. When there is a small amount of DNA present then it needs to be amplified with PCR prior to its analysis. VNTR cannot be used on old or degraded DNA and there needs to be at least several hundred base pairs present of intact DNA in the correct regions in order to conduct the analysis (Kluftinger, J. and Plunket, R., 2015). VNTRs were the first polymorphisms used in DNA profiling and they were successfully used in forensic casework for several years (The Council for Responsible Genetics). The use of VNTRs was, however, limited by the type of sample that could be successfully analyzed because a large amount of DNA was required (The Council for Responsible Genetics).
In this lab VNTR was used with the investigators DNA to determine if the crime scene DNA was contaminated with the investigator's DNA. When anybody enters a crime scene there is a risk that their DNA can contaminate the crime scene and this is why precautions are taken to ensure that the results that are discovered are true and accurate.
In this lab VNTR was used with the investigators DNA to determine if the crime scene DNA was contaminated with the investigator's DNA. When anybody enters a crime scene there is a risk that their DNA can contaminate the crime scene and this is why precautions are taken to ensure that the results that are discovered are true and accurate.
VNTR analysis protocol
Only very small amounts of DNA can be extracted from cheek cells at a time, PCR was used to amplify the DNA extracted.
To get DNA cheek cells were chewed and the mouth was rinsed with saline solution for 60 seconds. The cell saline solution was put into a centrifuge tube and microcentrifuged to create a pellet of cells at the bottom of the tube, more cell saline solution was added to create a sufficiently sized pellet. The cells were then re-suspended in lysis buffer. The lysis buffer contains beads, TRIS-HCl of pH 8.0, a chelating agent and proteinase K. The chelating agent removes Mg and proteinase K will digest any protein found in solution and leave DNA intact. The tube will cells and buffer was then incubated at 55°C for 15 minutes. The sample was then mixed and incubated at 100°C for 15 minutes. The solution was allowed to cool for 2 minutes then vortexed to mix. The tube was then centrifuged resulting in the DNA supernatant to be left.
The DNA supernatant was then added to a PCR tube containing a PCR reaction bead and D1S80 mastermix. The contents were then mixed and kept on ice until taken to the thermocycler and PCR will be started.
To get DNA cheek cells were chewed and the mouth was rinsed with saline solution for 60 seconds. The cell saline solution was put into a centrifuge tube and microcentrifuged to create a pellet of cells at the bottom of the tube, more cell saline solution was added to create a sufficiently sized pellet. The cells were then re-suspended in lysis buffer. The lysis buffer contains beads, TRIS-HCl of pH 8.0, a chelating agent and proteinase K. The chelating agent removes Mg and proteinase K will digest any protein found in solution and leave DNA intact. The tube will cells and buffer was then incubated at 55°C for 15 minutes. The sample was then mixed and incubated at 100°C for 15 minutes. The solution was allowed to cool for 2 minutes then vortexed to mix. The tube was then centrifuged resulting in the DNA supernatant to be left.
The DNA supernatant was then added to a PCR tube containing a PCR reaction bead and D1S80 mastermix. The contents were then mixed and kept on ice until taken to the thermocycler and PCR will be started.
DNA from the investigator's cheek was isolated, amplified and identified on a gel. The DNA that was amplified was from the D1S80 locus that has been mapped to the distal and of the 1p chromosome (Kluftinger, J. and plunket, R., 2015). The trait is inherited in an autosomal co-dominant fashion and is an example of a VNTR segment, in which the single 16bp sequence is repeated.
The DNA in this region is anonymous so it does not code for any particular proteins and it is not linked to any phenotypic traits but it is often used to distinguish a person's DNA fingerprint. This protects the confidentiality of the person(s) being investigated.
The DNA in this region is anonymous so it does not code for any particular proteins and it is not linked to any phenotypic traits but it is often used to distinguish a person's DNA fingerprint. This protects the confidentiality of the person(s) being investigated.