Basic Applied Bioinformatics

CHAPTER 20Primer Designing for SYBR Green Chemistry of qPCR

CS Mukhopadhyay and RK Choudhary

School of Animal Biotechnology, GADVASU, Ludhiana

20.1 INTRODUCTION

SYBR is a fluorophore that illuminates green after binding to double‐stranded DNA at the minor grooves. It is used in quantitative PCR (qPCR) to determine the amount of amplicon generated following each cycle of amplification. As real‐time PCR is very sensitive, and the presence of secondary structures and spurious amplicon would inflate the quantified amplicon, adequate care should be exercised while designing very specific and high‐quality primers for the SYBR Green chemistry of qPCR.

TABLE 20.1 Optimal and permissible ranges of parameters of qPCR primers (SYBR green chemistry).

SN	Feature	Optimal	Limits	Pros and cons
1	Length of each primer	21–23 nt	18–25 nt	Shorter primers could increase mispriming, and longer primers would reduce efficiency.
2	Amplicon	120–200 bp	100–220 bp	Primers for qPCR using chemistry other than SYBR Green use shorter amplicon (60–150 bp).
3	Primer Tm	64 °C	58–67 °C	Difference between the Tm of two primers should not be more than 2 °C.
4	Annealing temperature	60 °C	57–64 °C	The annealing and extension temperature of the Taq is recommended to be same.
5	GC% of primer	55–65%	35–80%	Higher or lower GC% would affect Tm of primers.
6	GC‐clamp at 3’‐end	1 clamp	Maximum 2 clamps	More than 2 G/C clamp with the last five bases at 3’‐end will make the primers sticky.
7	Run of ...