1
$\begingroup$

Why does one combine PCR and cloning as ways for amplification of sequences? Don't they produce the same result? I was reading the paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864885/ and got confused by the following passage:

Cloning and Bisulfite Genomic Sequencing

Each bisulfite-converted DNA sample was subjected to PCR by primers that did not discriminate between methylated and unmethylated sequences, using a GeneAmp PCR core reagent kit (Applied Biosystems). The primer sequences are listed in Table 1, and reaction conditions are listed in Supplemental Tables 1C and 1D (available online at http://ajp.amjpathol.org). Each subsequent PCR product was TA-cloned into pGEM-Teasy vector25 (Promega) for transformation into Escherichia coli strain JM109, according to the manufacturer’s instructions. Clones were picked randomly and colony PCR was then performed using vector primers T7 and SP6 to amplify the cloned inserts. Cycle sequencing was performed using BigDye version 1.1 (Applied Biosystems) and an automated capillary DNA sequencer Genetic Analyzer 3100 (Applied Biosystems). The sequences obtained were aligned and compared using SeqScape software (Applied Biosystems). The completeness of bisulfite conversion was first confirmed before scoring. The CpG sites sequenced as cytosine or thymine residues were scored as methylated or unmethylated, respectively. The methylated site frequency was calculated for each sample by dividing the total number of methylated sites over all cloned CpG sites.

Can someone comment on what purpose the cloning serves when the amplification is already done by PCR?

$\endgroup$
1
$\begingroup$

There are several reasons to clone a PCR product but the main reasons include expressing the product and maintaining the library inexpensively.

In the study you mentioned, the cloning and transformation is apparently done for obtaining single clones (from single colonies).

Clones were picked randomly and colony PCR was then performed using vector primers T7 and SP6 to amplify the cloned inserts. Cycle sequencing was performed using BigDye version 1.1 (Applied Biosystems) and an automated capillary DNA sequencer Genetic Analyzer 3100 (Applied Biosystems).

Since the original PCR would have a mix of different sequences, cloning is done to separate them out for the ease of studying them by sequencing. If they don't separate the sequences, they would have to use NGS (or more expensive methods like PacBio for longer reads). They have used Sanger sequencing. This article was published in 2007. At that time NGS was not as cheap as it is today and was hence not used by everyone. Also, the NGS machines of that time had even smaller read lengths (~40 for Solexa/Illumina GA and ~75 for GA-II). Considering these, Sanger sequencing may have been preferable to the authors of this paper. However, as I mentioned before, Sanger's method cannot be used for sequencing pooled samples; you need pure templates or else there will be mixed signals and base calling would be inaccurate.

(See the example chromatogram below: look at the position with the Y which has mixed T and C signals. It is not from a pooled sample, though).
enter image description here

Therefore cloning, transformation and colony picking was necessary.

Moreover, in many cases picking individual clones would be necessary for performing functional assays to associate phenotype with genotype.

$\endgroup$
  • $\begingroup$ Thank you for the answer. I am unclear about some points you made. Why would they need to use PacBio? The initial PCR product is still short enough for any short read sequencing method. I get the relationship of phenotype to genotype, but this is not the case here. Isn't the cloning just a step adding a bias to the methylation ratio quantification? $\endgroup$ – Cindy Almighty May 27 at 9:51
  • $\begingroup$ @CindyAlmighty You need to sequence single DNA molecules. That can be done using any NGS technology but not Sanger sequencing. However, for many of them the reliable read length is <300nt. With PacBio, you can sequence longer reads. Their reads are indeed smaller so they don't need PacBio. But, note that this article is from 2007. NGS was not that widespread and cheap at that time. There could be an amplification bias between methylated and unmethylated sequences but that would be there in the PCR product stage, itself. $\endgroup$ – WYSIWYG May 27 at 11:17
  • $\begingroup$ I understand the NGS vs Sanger vs PacBio. I am unsure how it figures in this problem - how is the molecule shortened by inserting cloning between two PCRs. $\endgroup$ – Cindy Almighty May 27 at 15:28
  • 1
    $\begingroup$ @CindyAlmighty it is not shortened. This paper is from a time when NGS was not common. Moreover, Sanger sequencing can give you up to 1kb of decent quality read. However, with Sanger, you cannot sequence a pooled sample. It would just cause interference. $\endgroup$ – WYSIWYG May 27 at 15:39
  • $\begingroup$ Oh, now we are getting on the right track I think. Your help is excellent, as it led me systematically to a solution. I think this is the exact point I was looking for - Sanger cannot sequence pooled samples. But now that I think about it, it is not interference, but we wouldn't be able to phase methylations. Do you agree? $\endgroup$ – Cindy Almighty May 27 at 21:42

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.