Advice 

What is meant by exonuclease activity?

Jeremy Anderson

What is meant by exonuclease activity?

Terminology: The ability to remove nucleotides one at a time from the end of a chain is called exonuclease activity. (exo = from the exterior or end).

Why do you need two primers in PCR?

Two primers are used in each PCR reaction, and they are designed so that they flank the target region (region that should be copied). That is, they are given sequences that will make them bind to opposite strands of the template DNA, just at the edges of the region to be copied.

Why primers are used in PCR?

​Primer. A primer is a short, single-stranded DNA sequence used in the polymerase chain reaction (PCR) technique. In the PCR method, a pair of primers is used to hybridize with the sample DNA and define the region of the DNA that will be amplified. Primers are also referred to as oligonucleotides.

Why is a buffer used in PCR?

PCR is carried out in a buffer that provides a suitable chemical environment for activity of DNA polymerase. The buffer pH is usually between 8.0 and 9.5 and is often stabilized by Tris-HCl. For Taq DNA polymerase, a common component in the buffer is potassium ion (K+) from KCl, which promotes primer annealing.

Which primer is most suitable for PCR?

Primers for PCR and sequencing should have a GC content between 40 and 60%, with the 3′ of a primer ending in C or G to promote binding. The 3′ end of the primer should be an exact match to the template DNA, because extension by DNA polymerase, during PCR, depends on a good match at the 3′ end.

Why do PCR primers have high GC content?

GC bonds contribute more to the stability—i.e., increased melting temperatures—of primer and template, binding more than AT bonds. Primers with 40% to 60% GC content ensure stable binding of primer and template.

What are the three main characteristics you need to consider for primer design?

It is important that a primer has the following characteristics:A melting temperature (Tm) in the range of 50 C to 65 C.Absence of dimerization capability.Absence of significant hairpin formation (>3 bp)Lack of secondary priming sites.