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Long-Range PCR (CAT#: STEM-MB-0211-WXH)

Introduction

Long-Range PCR is a method for the amplification of longer DNA lengths that cannot typically be amplified using routine PCR methods or reagents. For simple DNA templates, polymerases optimized for Long Range PCR can amplify up to 30 kb and beyond. For complex, genomic templates, 20 kb is a typical target.<br /><br />This method allows the amplification of more extended targets within a shorter period and with efficient use of resources.




Principle

Long-range PCR can be achieved by using modified high-efficiency polymerases with enhanced DNA binding, resulting in highly processive and accurate amplification of long fragments.

Applications

• Targeted resequencing and genotyping studies involving detection of gross alterations of the genome such as large deletions or substitutions.
• Genomic fingerprinting for screening of diseases.
• Validation of copy number variants (CNVs) in biomedical research.

Procedure

1.Primer design
2.Preparation of template
3.PCR reaction system and reaction parameters set
4.Detection of amplification products

Materials

1.fidelity Taq DNA polymerase: Vent polymerase (a thermostable archean DNA polymerase ), A combination of two different polymerases (one with high concentration, normally the Taq DNA polymerase and another one with low concentration, a high fidelity DNA polymerase.)
2.Primer:
The primers used in the long PCR are 22 to 30 nucleotides long and 50% of less GC content in it. Furthermore, it should have a low capacity of secondary structure formation and annealing temperature between 58 ̊C to 68 ̊C.