Question

This is also for the Unknown 3 I am doing for microbiology. If you could please help in detail thanks.

A. Now that you have confirmed that PCR product is of correct size, using agarose gel electrophoresis, how will you find out that the amplified DNA fragment is our desired target region?

B. What is the principle of Sangers’ sequencing technique? Use a labeled diagram (may copy & paste image from internet) to briefly describe the technique. Write two differences between the reaction mixes for PCR and Sanger sequencing. Name any other sequencing techniques that can be used to obtain nucleotide sequence of your PCR product.

C. Will you be able to identify your unknown organism(s) if you used DNA sequence for conserved regions (instead of variable regions) from your PCR product? (Yes or No)

Answer 1

A. In order to analyze whether the PCR product is the target region, the PCR product can be excised from the gel. In gel digestion is performed to extract the DNA in the extraction buffer. This DNA can then be sent for DNA sequencing using a Sanger’s sequencer. This sequence is aligned with the known sequence of target desired DNA to know whether PCR amplification was successful.

B. The Sangers sequencing technique uses dideoxynucleotides to interrupt the PCR. Wherever a ddNTP is added there is chain termination as there is no 3’ OH group on ddNTPs for Taq polymerase to add it to the newly synthesized strand. This will result in chain termination. Only one type of ddNTP (ddATP/ddCTP/ddTTP/ddGTP) is added to one of the four reaction tubes. The ddNTPs are labeled with different fluorescent dyes. Based on where chain termination occurs, fragments of different lengths are obtained. Image is attached.

These sequences can be aligned and the sequence can be deduced.

PCR	Sangers Sequencing
Only one product is formed of a fixed length determined by the PCR primers	Fragments of different lengths are obtained depending on where chain termination occurs.
ddNTPs are not used	ddNTPs are added and there is excess of dNTPs.
Both DNA strands are used for amplification.	There is linear amplification with only one DNA strand.
Detection is by Ethidium bromide method.	Fluorescence tags are used for detecting the fragments. The ddNTPs are tagged with fluorescent dyes.

Other sequencing method is the Maxim-Gilbert method in which the 5’ end of DNA is radiolabeled. Chemical degradation will remove single or 2-4 nucleotides in four tubes, by techniques such as hydrolysis, depurination etc. The sequence is then identified depending on fragments obtained on the gel by autoradiography. Nowadays next generation DNA sequencing methods are also used.

C. No.

Variable regions differ in nucleotide sequence between species. Conserved regions are the ones that will have same nucleotide sequence in all species of a genus and sometimes even in families. If variable regions are used for comparison, the species is easily identified. As conserved sequence will be same in many species, it will be difficult to identify the target organism. Hence, only variable sequence should be used.