CHAPTER 20
Primer 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 identical bases 2 bases Maximum 4 This will increase the possibility of secondary structure formation.
8 Proportion of different bases Equal percentage of each base Minimum 20–30% of one base __
9 3’‐end stability of primers Delta G > = –9 kcal/mol –7 to –11 kcal/mol Delta G of last five bases at 3’‐end should be higher (ignoring the symbol) than that of the internal region of the primer.
10 Concentration of Mg++ 3–4 mM of MgCl2 3–6 mM SYBR green buffer itself contains appropriate amount of MgCl2.
11 Max repeat mispriming 12 or more 11–15 Larger value inversely proportionate to the probability of repeat mispriming of the primers.
12 Check for template mispriming No mispriming Do not select primers with template mispriming Run Primer‐Blast of NCBI to identify the possible mispriming in the same species.
13 Number of unknown bases 0 0 No non‐specific bases should be present, to prevent non‐specific amplification.
14 Run of the same base 2 2–4 Run of the same base will increase the mispriming.
15 Checking the primer quality Delta G values should be higher for primer‐template and lower for primer secondary structures Identify potential hairpins and primer‐dimer/self‐dimer formation.

20.2 QUESTIONS

  1. 1. Design a pair of primers for SYBR Green chemistry‐based real‐time PCR of the TLR4 mRNA in Catla catla. Check the quality of the primers and evaluate them.
  2. 2. List the key parameters that determine the efficacy of qPCR primers (SYBR Green Chemistry).
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