Table of Contents
Pure DNA sample should be between 1.8 and 1.9 (the ratio of A260:A280). However, for this case, the DNA absorbance was 1.116 (A260/A230) at a concentration of 19.25µg/ml. This indicated contamination of the sample. Sample contamination can be one cause of the appearance of the faint band, leading to low DNA quantity (Bucalossi et al., 2011 p. 886). There are quite a number other reasons for faint bands (low DNA quantity). It may be due to high annealing temperature, the low number of PCR cycle, and low concentration of magnesium chloride.
The DNA quantity in 1 million blood cells may have undergone some form of shearing hence causing a low PCR product that gives a faint band. To solve the issue, each of the PCR components must be keenly checked for optimization. First, the programming for each step must be carefully considered due to an insufficient number of cycles, low annealing time, low extension time, low or high denaturation time, decreased denaturing temperature, and increased annealing temperature. Moreover, one may need to check primer concentration, dNTPs concentration, water impurity, polymerase concentration, and high GC rich templates. So this issue may be multifactorial, and should regard every directional aspect for PCR optimization.
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Lambda DNA was used in this lab to perform gel electrolysis. Lambda is a simple to use bacteriophage. It helps in determining and the number of DNA fragments. Lambda DNA contains DNA fragments with known size and can, therefore, be used for comparison with those of unknown fragments size in gel lanes (Reddacliff, Beh, McGregor, and Whittington, 2005 p. 435). Moreover, this kind of DNA can be used either cut or uncut into fragments by particular enzymes, for example, the enzyme DNA HindIII. Also, Lambda DNA size, as well as its commercial availability, makes it ideal for restriction analysis of DNA. The lambda DNA also acts on certain enzymes to produce the same output/results which should be observed in the DNA bands.
The similar size of DNA band in sheep blood is similar to the bacteriophage lambda DNA (Thomas, 2001 p. 8). This is because the lambda DNA has various recognition sites for various restriction enzymes and is commonly used to mark size, weight during the gel analysis of DNA. For this reason, the same size of the DNA band should be similar to the lambda DNA size. The HindIII and the marker lane should have the same cuts because they are constructed by the same elements.
The size of the PCR product was determined using the agarose gel analysis and was found to be in lane 4 (VR/VR). The size is approximately 10kbp. After the digestion of the DNA, the mutation gene would eliminate the sites for the restriction enzymes and form mutant genes/DNA molecules (Ryan, and Sweeney, 2005 p. 544). The sheep was observed to be resistance to Scrapie. The indications of Scrapie levels of resistance as per the genotypes are; AR (highly resistant), VR (resistant), AH (susceptible), and VH (highly susceptible).
The susceptibility of Scrapie is determined through the polymorphism of codons 171, 154, and 136. Codon 136 codes alanine (A) confers resistance to the protein that may structurally be changed to associate with Scrapie (Garcia-Crespo et al., 2004 p. 778). Also, the codon 136 confers to valine (V) associated with susceptibility. The PrP is, therefore, a genotype that helps to establish the resistance of sheep herds to Scrapie.
- Baylis, M. and Goldmann, W., 2004. The genetics of scrapie in sheep and goats. Current molecular medicine, 4(4), pp.385-396.
- Bossers, A., De Vries, R. and Smits, M.A., 2000. Susceptibility of sheep for scrapie as assessed by in vitro conversion of nine naturally occurring variants of PrP. Journal of Virology, 74(3), pp.1407-1414.
- Bucalossi, C., Cosseddu, G., D’Agostino, C., Di Bari, M.A., Chiappini, B., Conte, M., Rosone, F., De Grossi, L., Scavia, G., Agrimi, U. and Nonno, R., 2011. Assessment of the genetic susceptibility of sheep to scrapie by protein misfolding cyclic amplification and comparison with experimental scrapie transmission studies. Journal of virology, 85(16), pp.886-892.
- Elsen, J.M., Amigues, Y., Schelcher, F., Ducrocq, V., Andreoletti, O., Eychenne, F., Khang, J.T., Poivey, J.P., Lantier, F. and Laplanche, J.L., 1999. Genetic susceptibility and transmission factors in scrapie: detailed analysis of an epidemic in a closed flock of Romanov. Archives of virology, 144(3), pp.431-445.
- Garcia-Crespo, D., Oporto, B., Gomez, N., Nagore, D., Benedicto, L., Juste, R.A. and Hurtado, A., 2004. PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR. The Veterinary record, 154(23), pp.717-722.
- Lühken, G., Buschmann, A., Groschup, M.H. and Erhardt, G., 2004. Prion protein allele A136H154Q171 is associated with high susceptibility to scrapie in purebred and crossbred German Merinoland sheep. Archives of virology, 149(8), pp.1571-1580.
- Reddacliff, L.A., Beh, K., McGregor, H. and Whittington, R.J., 2005. A preliminary study of possible genetic influences on the susceptibility of sheep to Johne’s disease. Australian veterinary journal, 83(7), pp.435-441.
- Ryan, M.T. and Sweeney, T., 2005. Genetic susceptibility to scrapie in sheep: a clinically relevant theme in veterinary medical education. Journal of veterinary medical education, 32(4), pp.544-550.
- Thomas, D.L., 2001. Genetics Of Scrapie Resistance In Sheep. AUGUST 25, 2001, p.8.
- Thuring, C.M.A., Erkens, J.H.F., Jacobs, J.G., Bossers, A., Van Keulen, L.J.M., Garssen, G.J., Van Zijderveld, F.G., Ryder, S.J., Groschup, M.H., Sweeney, T. and Langeveld, J.P.M., 2004. Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein. Journal of Clinical Microbiology, 42(3), pp.972-980.