Q.1.
To determine the solo-LTR region I first ran an electronic PCR on s240c3, s165c5 and s399c8 primers. I then aligned the PCR products to each other using a series BLAST searches, identifying a related sequence of ~968bp expected to be the same in each PCR product. Using BLAST I identified the start and end points of this sequence in relation to each of the PCR products of the 3 primers used in the experiment, allowing me to determine the direct repeats and thus pick out the Solo-LTR sequence and pre-integration site. From the size of the solo-LTR sequences, the size of the whole sequence and the identification of the direct repeat sequences I was able to work out the size of the preintegration site by taking the whole PCR product size and eliminating the size of one of the direct repeats and the entire solo-LTR sequence. The results are as follows:
s399c8=A
1558bp
968 = Solo-LTR size 584 = Pre-integration site size
s165c5=C
1204bp
967= Solo-LTR size 231 = Preintegration site size
s240c3=E
1269bp
968= Solo-LTR 295 = Preintegration site
Q.2.
Figure 1 Result of gel electrophoresis of my DNA sample
The product of the reaction of the A primer seems to have failed as no bands were produced apart from the terminal point of the migration which is too small to be considered as either a preintegration site or a retrovirus containing section, not only did my partner seem to have the same problem, most if not all of the submitted gels seem to have no bands for the A set of primers.
For the E primer sets, there are two strong bands. Around the 1018 MWM bands which approximately cor...
... middle of paper ...
...cluded here but ~15 were used in the search) apart from the last base on the direct repeat, so the sequence is unlikely to be totally irrelevant, it may represent a retro-element that the solo-LTR was derived from.
Q.6.
One method of identifying novel solo-LTRs within an individual would be to sequence their genome (pyrosequencing makes this somewhat viable) and align it to the current human genome sequence using NCBI blast, “jumps” may be noticed at certain point (i.e. a section which starts at base 260 in the human genome sequence but 960 in the individual sequence) the insert is likely to be due to a large retro element integration event, solo-LTRs can be easily confirmed by identifying direct repeats. You can then use knowledge of the flanking sequences to produce PCR primers to test for it in others.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
it is of medium intensity. Figure 1. Atoms labelled in 3 used in the NMR assignment. The 1H NMR spectrum shows that there are 18 protons in 11 different proton environments.
The ligation was expected to make four combinations. The original pBK-CMV and CIH-1 fragments would region to make a non-recombinant pBK-CMV/CIH-1 plasmid. The original pUC19 fragments would rejoin to make a non-recombinant pUC19 plasmid. The larger fragment of pBK-CMV and the small 27bp fragment of pUC19 or the desired recombinant vector, CIH-1 fragment and the larger 2659bp pUC19 fragment. As pBK-CMV does not contain the ampicillin gene then transformed Ecoli containing these would not to survive on the Agar leaving only pUC19 recombinants and non-recombinants.
...rrier. There are available tests you can take to determine the possibility of your children receiving the disease.
Since one of the prominent concerns she has is related to health, she needs to be reassured by a physician that these symptoms are not dangerous, along with being aware about the fact that she misinterprets these symptoms and these symptoms can be created if she persistently focus on the certain parts of her body.
Polymerase Chain reaction (PCR) is used to isolate a predetermined strand of DNA on the double helix. Once the desired DNA is isolated it is able to be copied as much as needed (2). In this experiment PCR was used to isoloate Vangl2 from Zebra fish embryos. In a PCR experiment, a primer is used to find and isolate the desired nucleotide sequence of DNA (2). In this experiment two primers were used as follows:
Mental health is being aware, accepting yourself, and striking a balance in all aspects of your life like social, spiritual, physical, economical, and mental (Association, 2001). Mental health can be described as our positive interactions with the context and events in our life, and having the ability to cope with life’s stressors. Mental health problems can begin at anytime during your life (CAMH, 2010). In fact anything can make it difficult for an individual’s ability to interact effectively, and may lead into a mental health problem (Association, 2001).
The term ‘dual diagnosis’ refers to people who suffer from grave mental illness and have problems with drugs or alcohol to the extent that their mental and physical health is affected. The condition of substance misuse disorder does not entail that there is dependence or an addition rather it defines a spot where the person’s use of drugs or alcohol has become problematic and it impairs the person’s tone of spirit and their ability to work as part of a community. Some reasons that people who are mentally ill drink and get hold of drugs include they are self-medicating, to normalize entry into social groups, to run away or to disengage because their spirit is difficult so they why would rather be “numb” than deal with their troubles. In this paper I will cover the following topics substance abuse’s role in offending behaviors, challenges for both client and clinician’s perspective, interventions and techniques that can be used with this population and some research findings.
Modern techniques , rather than the gene map , maps the map of the DNA within the gene itself : the positions of short sequences " marker " are used as markers signaling over the cromosssomas . Once a gene is discovered, it is necessary to unravel its base sequence prior to its function being studied . The sequencing has become easier with the development of methods for cloning the DNA - producing large amounts of identical fragments. In the method most widely used DNA sequencing , the chain is denatured into single strands . These are then used as templates for DNA synthesis , but such that replication to as the double helix reaches a certain growth in the mold base . In addition to provide DNA polymerase and the four bases, A - G -C- T, also using small amounts of these dideoxynucleotide bases. This is incorporated , as the normal bases, the double helix growth but prevent the continuation of the chain. The fragments are then separated by gel electrophoresis and the base seq...
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
Caplan M. Geralyn. DNA Isolation Lab. Owensboro Community & Technical College, n.d. Web. 5 April 2014.
The reason for this pattern is the same as that that was made in the
...nal peaks in the spectrum are at much higher wavenumbers because of the lighter elements involved. The C-H stretch is at 2976.58 cm-1 and the N-H stretch is at 3265.02 cm-1. These peaks all correspond well with the literature values2.