Question

Identification of unknown Bacteria by sequencing rDNA

Having a little trouble understanding the PCR process. This is a lab we did and some homework questions regarding the sequencing rDNA to find unknown bacteria. I hope by answering these I can have better understanding of the process. The more descriptive the better.

Thank you!

2.

A. List the reagents used in preparing master mix for PCR and write one sentence about why each one was necessary.

B. 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?

C. 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.

Answer 1

A.

Taq DNA polymerase- to add nucleotides i.e., to grow the sequence

dNTPs- it gets added to the primer

MgCl_​​​2 - acts as a cofactor and catalyst of the Taq DNA polymerase .

Reaction buffer - to create optimal conditions for the Taq DNA polymerase to function.

B . PCR amplifies specific sequences only.

Other than this we can also use affinity chromatography to be sure if the amplified product is our target region.

C. In Sanger sequencing method, dideoxy nucleotides are added along with deoxynucleotides. Due to the presence of ddNTP, no more nucleotides are added after resulting in the end of the polymerase reaction. So we get different sizes of bands which helps us in sequencing genome.

Reaction mixture : dNTP, polymerase, Mgcl2, buffer , ddNTP