Sunday, 22 November 2009

GGS LIVE - Cloning

Method: Cloning

About: What is cloning you ask! I see cloning as set of methods which allow to copy specyfic piece of DNA and then to use it as we wish. 

What: Amplification of three different cDNAs (cDNA is a protein coding part of the gene for more informations see this amnimation McGraw Hill Animations - cDNA (complementary DNA)) by PCR and transfering them (cloning) into first transient vector and afterwards to a final (target) vector. Each of our cDNAs is around 300bp.

How: by series of DNA digestions and ligations.

Before we start please have a look at this cool animation about the cloning:) McGraw Hill Animations - Steps in Clonig a Gene

When we start cloning from the scratch first what we have to do is to amplify the desired DNA sequences using the PCR technique (about the PCR in the future Biochemistry Methods Section:). After amplification, sample of the reaction (around 10%) is analysed by agarose gel electrophoresis to check presence of the desired product. Example of such gel you can see on the Figure 1.

You can see here three different products of PCR (each around 300bp, compare to the ladder DNA) which from now on we will call S1, S2 and S3. Positive Control lane is a PCR product that previously was shown to be ampliefied (we used that as a validation of our PCR) and in the Negative Control lane no DNA tamplate was used (no PCR product is expected). After we make sure that our products are fine (and they are, all of them have expected size, PCR worked ==> Postive Control) we need to prepare another gel where we separate rest of the reaction volume (95% that left) and extract/purify the desired DNA band from the gel. Look at the Figure 2:)

After extraction purity of the DNA is analysed (again by running the gel) and then DNA is ligated/fused with  transient vector. Vectors used in this step are designed to facilitate the fusion of the PCR product with the plasmid. In our case we will use a vector that is provided in linearised form, ready to go:) See Figure 3.


As you can see size of our vector (which we are going to call pXT) is around 3,0 kb (3000bp). After the ligation, bacterias (usually the E.coli) are used to multiply the plasmid so we will have a good amount to work with. After plasmid DNA isolation out of the bacterias we need to test if the ligation took place, so we perform a control DNA digest. See Figure 4.


From the different restriction enzymes available (I mean those that cut our plasmid and insert DNA) we chose those that will give us the simpliest (or the most obvious) band pattern. Example above:) I choose BamHI enzyme that cuts once in the backbone of the vector and once in our DNA of interest (all three cDNA's are similar sizes and have the single BamHI site so I did not show the other two). This way digest of the DNA should give us two bands, if the ligation took place and only one band (around 3,0kb), if the ligation failed.
After we make are sure that we have our DNA ligated into transient vector, we might either sequence the DNA to check if it is what we expect (mutations can be introduced during PCR reaction) or go to the next step (it depends on what we are going to do with the DNA after the cloning).
In case of the cDNA it is common to get it sequenced. When we get the sequence results back and everything is ok we go to the next step which is to clone the insert DNA of interest from transient vector to the final vector:) In order to do that we digest the target vector (pXA, size around 5,5kb) and cut the inserts out of the pS1/S2/S3 vectors with the same restriction enzyme mix. Remember that each restriction eznyme generates different DNA ends. To make our live easier, we design cloning in the way that we can use the same restriction enzymes to cut the vector and inserts out. This way we generate DNA ends that are compatible what increase efficiency of the ligation step. See Figure 5.

On the left side of the Figure 5 we see our target vector (size 5.5kb) and our transient vectors (pS1/S2/S3) bearing the inserts. We digest them with HindIII/XbaI enzyme mix to linearize target vector and to cut our inserts out like you see above. In the next step we have to extract the insert DNA from the gel to separate it from the backbone. Afterwards we ligate the inserts with the target vector. Then using bacterias we amplify DNA and again after isolation of plasmid DNA we set up the control digest to see if we got it ligated (this time to the final vector). Check it out on Figure 6.

This time I choose a restriction enzyme PvuI that cuts three times in the backbone of the pXA vector and either once or does not cut at all in the insert DNA. As you see depending on the insert sequence the PvuI site is in different position what results in specific band pattern for each of the vectors. Please notice that bands 1069bp and 1096bp will appear as one big band as they do not seperate greatly (their size is too similar).

This is it:) After we make sure that our plasmids are ok (we can perform another control digests) we can finally use our DNA. About that in the future Biochemistry Methods Section.

Cya Soon:)

Maciek GGS Team

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