PrimeSTAR LongSeq DNA Polymerase has extremely high specificity, making it possible to amplify ultra-long chains of over 50 kb and amplify difficult sequences such as long GC/AT-rich sequences.  This high specificity requires primers have a melting temperature (Tm value) of 70°C or higher, greater than what most general PCR enzymes require. To increase the Tm value of primers, we recommend extending the 5' end of the primer to increase its length, therefore ensuring sufficient amplification.

• Use ~35-mer primers with a Tm value >70°C, preferably with a Tm of 74°C or higher
• If the Tm value is below 70℃:
  • Use a 3-step PCR protocol that allows for use of an annealing temperature that differs from the recommended 68°C extension temperature
  • Lower the annealing temperature by 5℃ or more below the Tm value 
• Match the Tm values of primers in multiplex PCR
• Use of a phosphothioate-modified base at the 3' end may improve amplification 

To demonstrate successful amplification using the above primer design specifications with PrimeSTAR LongSeq DNA Polymerase, we designed primers of varying lengths, ranging from 15–34 bp in length (Table 1), to amplify a 24 kb region of the β-globin gene.

PCR reactions were set up according to the PrimeSTAR LongSeq DNA Polymerase Data Sheet, using 100 ng of human genomic DNA as the input template. A Clontech PCR Thermal Cycler GP (Cat. # WN400) was used for either 2-step or 3-step PCR cycling conditions, with annealing temperatures of 68°C and 57°C, respectively (Figure 1). A 3-step PCR protocol was used to demonstrate the effect of changing the annealing temperature on successful amplification.

Following the PCR reaction, products were run on an E-Gel. A 1 kb Plus DNA ladder was used to mark sizes on the E-gel.

For 2-step PCR cycling conditions, specific amplification of a 24 kb long chain was confirmed when the annealing temperature was set to 68°C and primers with a Tm value of 69°C or higher were used (Figure 2, Panels A and C). Amplification was not observed for primers with a Tm value of 62°C or lower because the annealing temperature of 68°C used in the 2-step protocol is not 5°C below the primer Tm value. 

For the 3-step PCR cycling conditions, specific amplification of a 24 kb long chain was confirmed when the annealing temperature was set to 57°C and primers with a Tm value of 62°C or higher were used. A 24 kb long chain amplification was not confirmed with primers having a Tm of 56°C (Figure 2, Panels B and C). 

Due to the high specificity of PrimeSTAR LongSeq DNA Polymerase, we recommend primers with a Tm value of 70°C or higher to ensure sufficient amplification of your target sequence. The Tm value of primers can be increased by extending the 5' end of the primer to increase its length. If the Tm value of primers is lower than 70°C, a 3-step PCR protocol may be used. Designing primers with a Tm value of at least 70°C will ensure sufficient amplification of your ultra-long, GC/AT-rich sequences.