Molecular Genetics - Report Example

MOLECULAR GENETICS PRACTICAL REPORT Question1 Annotation of the Gel picture Lane This lane contains the undigested plasmid. Lane 2- This lane shows the HindIII digested product which maybe linear. Lane 3- This lane shows the lambda DNA digested with HindIII. Lane 4- This lane shows the ligated product. DISCUSSSION For the practical session we isolated plasmid and ran them on an agarose gel. Gel electrophoresis separates DNA fragments by their size or molecular weight. The agarose gel acts like a sieve, separating different sized fragments while the electric current provides the driving force. On the gel the circular form of the nucleic acid would move a greater distance from the gel as compared to the linear form .This is because the movement of nucleic acids through the gel depends on the molecular size as well as the shape of the nucleic acid molecule. The more extended the molecule, the more resistance it faces, and so the velocity of the linear form on the gel is decreased as compared to the circular form. The circular form moves faster on the gel as because of its compactness and thus faces a lesser frictional force. After this, we calculated the size of the plasmid using a molecular ladder. With the help of a molecular ladder which can be obtained by digestion of the λ DNA with HindIII, the size of the plasmid isolated can be approximated. Lambda DNA on digestion with HindIII breaks into seven fragments of sizes 23.130 kb, 9.416 kb, 6.557 kb, 4.361 kb, 2.322 kb, 2.027 kb and 0.564 kb. As because the concentration of the digested lambda DNA was less all the digested products could not be visualized i.e. only three bands of the digested DNA which was of larger size could be visualized whereas the smaller fragments could not be visualised. Comparing the band of the plasmid isolated and the bands obtained by the lambda DNA digestion (molecular ladder) it can be approximated that the size of the plasmid isolated is around 3.5-4 kb as the band of the isolated plasmid is further down the third band obtained and would have corresponded to a region between the fourth and fifth bands by digestion of the lambda DNA. The other who ran the gel using the plasmid that they had extracted also estimated the size of the plasmid using the same procedure. The size of the plasmid isolated by Group H can be analysed by the same procedure. The plasmid isolated by Group H corresponds to the third band of the lambda digested product can be approximated to a size of around 6kb. After digestion of the plasmid, two bands were obtained indicating the presence of 2 sites for HindIII. The lambda fragments joined on treatment with ligase to form a larger molecule of size of around 48.337kb. The ligation procedure was successful as it is very well visualized by comparing the mobility of the ligated product with that of the fragments. The ligated product being larger in size moves with a lesser velocity. The gel that we used for the procedure was made of agarose and it was dull in appearance as it does not fluoresces (glows) and it provides a perfect background for the visualization of the DNA bands which fluoresces. The DNA which was loaded in the well was found to move towards the positive end of the electric field. However, the final result was not something that I anticipated. The gel was expected to show the presence of the DNA fragments in the form of bands. These bands would be located at regions where the DNA molecules migrate (towards the positive end of the electric field) under the influence of the electric field depending on the size as well as on the shape. The gel showed the above expected results. However, the bands were expected to be distinct but instead indistinct bands and lots of contamination of protein was obtained in the lanes of lambda digested products and lambda ligated products. Moreover, certain extra bands were obtained on the lanes of both digested as well as undigested plasmids indicating the presence of RNA as a contaminant. These RNA contamination can be got rid off by treatment of the isolated plasmid product with RNase (ribonuclease). To sum it all up, the bands we obtained were not standard due to contamination. QUESTION 2 A) The number of recognition sites that the plasmid has for HindIII is two. The number of recognition sites for Pst1 is three. B) Restriction map It is given in the question that HindIII cuts two fragments- 2.9 Kb and 2.1 Kb. According to the hint in the question, one of the sites is at position ‘0kb’ in the circular DNA thus when cut at position ‘0kb’ we get a linear DNA of size 5kb. Since we get two fragments the other position must be at position 2.9 kb. Now, the double digested product of HindIII and Pst1 together gives five fragments-1.8 kb, 1.5 kb, 0.7kb, 0.6kb and 0.4 kb. Since the fragments 1.5kb and 0.6kb sums up to 2.1 kb there must be a cutting site of Pst1 at position 3.5kb. Again, the fragments of size 0.7 and 0.4 after double digestion indicate the presence of a cutting site of Pst1 at 0.7kb position and 1.1 kb position. Now, to verify by seeing the restriction map obtained by digestion of the plasmid with only Pst1. We can see that the fragment between 3.5 kb and 0.7 kb (taken clockwise) is 2.2kb. The fragment between 0.7kb and 1.1kb (clockwise) is 0.4kb in size while the fragment between 1.1kn and 3.5 kb (clockwise) is 2.4 kb in size. Thus the addition of all the fragments make up the 5kb circular DNA. c) To decide which of the possibilities is correct I would perform the following experiment named Restriction Mapping: The plasmid is first treated with HindIII and the digested product is run on an agarose gel. The two fragments obtained are visualized as two bands corresponding to sizes 2.1kb and 2.9kb. The region of the gel containing these bands is to be carefully cut and the two gel strips thus obtained is to be placed separately in two different dialysis tube containing buffer. Then the process of gel elution is to be carried out which would result in the elution of the plasmid fragments out of the gel and into the respective buffer containing dialysis tubes. The plasmid fragments thus obtained is to be carefully collected and treated in different vials with Pstl and the double digested products are again gel electrophoresed. It will be observed that the plasmid fragment of size 2.1kb will be further digested by Pstl into two fragments (and thus give two bands)of sizes 1.5kb and 0.6kb.While the other plasmid fragment of size 2.9kb will be digested further by Pstl into three fragments (three bands) of sizes 1.8kb,0.7kb and 0.4kb. Thus it can be stated that the plasmid fragment obtained by treatment of HindIII of size 2.1kb has only one cutting site for Pstl corresponding to position 3.5kb of the intact plasmid whereas the plasmid fragment of size 2.9kb has two cutting sites for Pstl corresponding to positions 0.7kb and 1.1kb. Question 3 Polyacrylamide gels are widely used to separate proteins, small RNA molecules and very small DNA molecules. However, most DNA molecules used in molecular biology have molecular weights that are so high that they fail to penetrate even a weakly cross-linked Polyacrylamide gel so agarose gels are used instead. A 0.8% agarose gel can accept DNA molecules of as large as 50x10^6. DNA molecules are negatively charged by virtue of the negatively charged phosphate groups. When the DNA molecules are subjected to an electric field it migrates toward the positive end of the electric field. This movement of DNA molecules depend on the size as well as on the conformation of the molecule. Larger sized DNA molecules take longer time to migrate the same distance as that traversed by a smaller size molecule as the former faces more restriction to pass through the sieves of the agarose gel. The resolution of DNA fragments on the basis of molecular weight is extraordinary as molecules differing by as little as 1% in their molecular weight can be separated. Furthermore the range of molecular weights of DNA to be separated ranges from 10bp to 3x10^5bp. However, for different size ranges different gel concentrations should be used as very large molecules would not be able to pass through the more concentrated gels and very small molecules would pass through the very dilute gels as such that their velocity would not depend on molecular weight. The relation between the distance(S) and molecular weight (W) is: S=a - b logW Where “a” and “b” are constants that are functions of duration of electrophoresis, buffer and the concentration of the gel. Moreover the ability of a DNA molecule to pass through the gel also depends on the shape of the molecule. For example a linear, rod-like molecule encounters greater resistance than the DNA molecule of the same size but in the form of a dense sphere. Thus there is a difference in the mobility of linear, circular and super-coiled forms of a DNA molecule. However it is difficult to predict the relative mobility of the different forms of DNA as it is not only the frictional coefficient that is important but also the flexibility of the molecule. For example, a linear molecule has a higher mobility than a more compact nicked circle as the free ends of the linear form can easily dodge their way through the gel as compared to the single nicked circular form. At the same time a super coiled form has more mobility than a nicked circle as it is much more compact than the latter. However the compact super coiled form is not as rapid as the linear form of DNA. Moreover the nicked circle has the same flexibility as that of the non-super coiled covalent circle but when a twist is introduced in the latter its mobility increases. So DNA molecules of different sizes (or a mixture of DNA fragments) and conformation can be separated using the technique of Agarose gel electrophoresis. REFERENCES Serwer,P.(1983). Agarose Gels: Properties and uses for electrophoresis. Electrophoresis, 375-382. Zwaan, J., 1967. Estimation of molecular weights by polyacrylamide gel electrophoresis. Anal.Biochem, 21, 155-168. Freifelder, D., (1999). Physical Biochemistry. 2nd ed. Ny: W.H.Freeman & Co. Wayne, M.B, 1994. PCR amplification of upto 35kb DNA with high fidelity and high yield from Lambda bacteriophage templates. Proc.Natl.Acad.Sci.USA, 91, 2216-2229. Csus.edu. 2010. lambda HINDIII Ladder. [ONLINE] Available at: http://www.csus.edu/indiv/r/rogersa/sizemarkers.pdf. [Accessed 17 January 13]. Smith, R., 1976. General principles and Applications to Agarose Gel Electrophoresis. Clinical Chem., 22, 497-499. Birnboim, H.C., 1983. Rapid alkaline extraction for isolation of Plasmid DNA. Methods enzymol (UA), 100, 243-255. Hinnebusch, J, 1993. Linear plasmids and chromosomes. molecular microbiology, 10, 917-922. Kado, C.I., 1981. Rapid procedure for detection of small and large plasmids. Journal of Bacteriology, 145, 1365-1373. Brammar, W.J, 1980. A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes.Gene, 10, 93-99. Read More