[问答题]
The cloning of plasmids is an essential skill for man
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dekw-w ,9wp,u There are various techniques for this type of cloning, but the first steps involving the u
dp,w, w-9ukewse of restriction endonucleases and DNA ligation are long and often inefficient. The development of more reliable and economical techniques has been important in increasing our ability to manipulate DNA.
In vivo techniques have been successful in yeast and E.coli using DNA fragments with overlapping nucleotide sequences. However, if the overlapping sequences are too long it becomes problematic and sometimes more expensive because of the size of the primers required. To try to increase the efficiency of this method, further research was carried out, using E. coli and DNA fragments with homologous ends prepared using the PCR technique from template plasmids using Q5 DNA polymerase. In the first experiment, 2.6 kB plasmids were formed inside E. coli from 2 DNA fragments with varying lengths of overlapping nucleotide sequences (nt OL). Successful recombinations could be identified, as only these plasmids led to the fluorescence of the cells.
1.Describe the pattern of successful recombinants for the E.coli colonies shown in the photographs.
2.What is the function of DNA polymerase in the PCR technique?
The cloning accuracy was calculated as the percentage of the total colony number that was fluorescent. The total number of colonies and the cloning accuracy for the different overlapping nucleotide lengths are shown in the table and graph below.
3.Calculate the number of colonies with successful DNA recombinations on the agar plate with 25 overlapping nucleotides.
4.1.Explain how DNA recombination occurs with overlapping nucleotides.
4.2.Suggest a reason for the results seen in the agar plates containing E.coli with DNA fragments with 0 or 6 overlapping nucleotides.
5.Suggest a possible reason for the high cloning accuracy with 9 overlapping nucleotides.
Cloning efficiency was then investigated with different numbers of DNA fragments which had 18 or 25 overlapping nucleotides. The results from the experiment are shown below.
6.From the photographs of the petri dishes, deduce what effect the number of DNA fragments has on the number of colonies with successful DNA recombinations.
7.Calculate the percentage increase in the number of colonies for the 25 nucleotide overlap compared to the 18 nucleotide overlap for the recombination of 2 DNA fragments.
The investigation was extended to see how plasmid size and the number of DNA fragments affected DNA recombination in E.coli. The graphs below show the colony numbers resulting from either 2 DNA fragments or 3 DNA fragments being joined together to form different-sized plasmids (measured in kilobases).
8.Compare and contrast the results for the number of colonies produced from 2 DNA fragments and 3 DNA fragments.
Lastly, the accuracy of the cloning of the plasmids in colonies was tested. Colonies containing the 3 different sized plasmids were selected at random, plasmids were extracted and these were tested using gel electrophoresis. The results of 6 plasmids from each of the 3 different sizes are shown below.
9.Identify the plasmid number and size that has not been cloned accurately.
10.Using all the results provided, discuss whether they support the hypothesis that the most efficient process for bioscience research using plasmid recombination and cloning in bacteria is to use 2 DNA fragments, with 25 nucleotide overlap to form 2.6 kB plasmids.
