Background: You are working in a biochemistry/molecular biology lab that special
ID: 146078 • Letter: B
Question
Background: You are working in a biochemistry/molecular biology lab that specializes in protein applications (purification, identification, etc). The lab has been working with a new organism. This organism is single-cellular and eukaryotic and appears to be related to yeast. You are entering the lab and have been assigned characterizing this new organism.
PCR and Cloning 7. How might you use PCR to isolate DNA fragments from the organism? What about isolating mRNA? 8. What plasmids would you use for question #7? what properties are of importance to plasmids? How would you prove that you inserted DNA into the plasmid? (Note this also relates back to Chapter 15 with Recombinant Proteins)Explanation / Answer
7)
1. Isolating DNA- To isolate the dna first we need to remove the cell wall using any one of these- cellulase, chitinase, lysozyme according to the organism.
Then we apply RNAse and protease to remove rna and protein respectively. After this chilled ethanol is added and DNA precipitates out as fine threads.
2. Cutting of DNA- Restriction.enzymes are used to cut the dna. Agarose gel electrophoresis is applied to check the progression of cut dna fragments.
3. Ligating- The vector dna and host dna which are cut using the same restriction enzyme are joined by dna ligase.
4. Amplification using PCR- multiple copies are synthesized using two sets of primers and dna polymerase enzyme. It is performed in three steps:
a) Denaturation- the temperature is increased to 94-98 degree Celsius , this breaks the hydrogen bonds between the two strands of dna.
b)Annealing- The dna is cooled to 50-65 degree Celsius. Primers are added and the dna is allowed to anneal to the two primers.
c) Extension- dna polymerase is added to the mixture to initiate the synthesis of two new chains complimentary to original dna chains. A thermostable polymerase called taq polymerase is used.
We cannot usePCR to amplify rna directly as Taq polymerase do not work on rna. However the enzyme reverse transcriptase along with PCR can be used to isolate m-rna. This technique is known as RT- PCR.
8)
The plasmids used are yeast integrative plasmid and yeast duplicative plasmid.
The properties important to plasmids are -
a) origin of replication- This is a sequence from where replication starts.If we want to receive high copies of the dna then it should be cloned with a plasmid having high copy number.
b) Selectable Markers- It is used to distinguish between recombinant and non recombinant DNA. The genes for antibiotic resistance such as ampicillin, tetracycline are important selectable markers.
c) Cloning sites- Vector needs to have few recognition sites for the commonly used restriction enzymes. Else it will be cut into too many parts.
That dna is inserted into plasmids can be proved by the following ways-
a) The ligation of alien dna is carried out at a restriction site present in one of the antibiotic resistance genes.for eg.- if gene is ligated at the tetracycline resistance gene then it leads to insertional inactivation of tetracycline gene. If the transformed cell( a cell having recombinant DNA) is placed in a medium containing tetracycline then the cell will not grow. This proves that the cell has recombinant DNA.
b) Another method is using alternate selectable markers which produce colour if the cell's dna do not have an insert. eg. If the dna is inserted into the coding region of beta galactosidase then it will not give any colour. If there is no insert then it will give blue colour.
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