Long PCR, also known as long range PCR, long and accurate PCR, is a technique that could amplify longer DNA fragments such as 5kb, 10kb or 20kb DNA fragments. Some of the mutation detection techniques depending on the fragment size of the DNA, for example, the Fragile X syndrome. The fragment of such a DNA is too long, so a long PCR reaction is required. Compared with conventional PCR, long PCR needs higher concentration of reagents, long cycles with extended time and extreme precision and expertise. Long PCR can be applied in genotyping, DNA fingerprinting, DNA sequencing of long fragments and copy number variation studies. The advantages of Long PCR is that specificity of the reaction is increased and it can amplify longer DNA fragment. Long PCR is the first choice for the diagnosis of long CpG repeat-containing templates. It is also very useful in downstream application.
Fig. 1 Comparison between long PCR and conventional PCR.
To achieve long PCR, a different type of Taq DNA polymerase, different types of primer and PCR reaction buffer are needed. The normal Taq DNA polymerase doesn’t work here because of low fidelity and low proofreading activity. It can add dNTPs up to 1kb to 2kb, and the chance of the incorporation of the wrong dNTPs are increased because the specificity of the Taq will gradually decrease. The 3′ to 5′ exonuclease activity is essential for long PCR. Two kinds of DNA polymerases are used in the long segment PCR. One is the thermostable DNA polymerase (Taq enzyme), which is used in the conventional PCR. It has strong extension ability and depends on this enzyme for chain extension. The other is a thermostable DNA polymerase with 3′→ 5′ exonuclease activity (commonly used Pfu enzyme), which has a good function of proofreading and can cut the mismatched base and reuse the first enzyme for chain extension. The two enzymes have their own advantages and disadvantages. By making full use of the extension ability of the first enzyme and the reading function of the second enzyme, the two enzymes complement each other in the reaction and complete the amplification of long DNA fragments.
The primers used in the long PCR reaction must be also different from the normal PCR reaction. The primer used in long PCR is 22 to 30 nucleotide long, contains more than 50 % of GC, lower secondary structure and has annealing temperature between 58 ℃ to 68 ℃. Use primers from 0.1 to 1.0 mM as required. Theoretically, a primer concentration of 100 pM to 200 pM is used for fragments up to 20kb. In case of complicated PCR template DNA use, we use low concentration of primers. If simple or lower complicated template DNA uses, a high concentration of primers is used.
In terms of buffer, the long PCR Reaction buffer should contain a high concentration of MgCl2. The concentration of magnesium ion in long PCR depends on the purpose of reaction, since a higher concentration of reaction increases the non-specific bindings into the reaction. The content of MgCl2 in buffer must be 2mM to 5mM MgCl2.
(1) Cloning Long DNA Fragments
Because eukaryotic genes have introns and exons, the genes are generally longer. The conventional PCR technology is difficult to amplify the full length, and long PCR provides a technical platform for the amplification of such genes. The ability to amplify fragments as large as 20 to 50 kb will make it easy to isolate complete genes from cDNA, thereby eliminating the need for time-consuming screening of target genes from genomic libraries.
(2) Genomic Research
In the study of genomic diversity, the key role is the complex of polymorphic markers on chromosomes-single-mode specimens. This marker is useful for mapping disease-related genes and analyzing knockout mice.
(3) Detection of Mutations and Gene Expression
Long PCR technology is widely used in the detection of gene mutations and gene deletions. A large mutation in the TSC2 gene is one of the causes of autosomal dominant hereditary nodular sclerosis (TSC). The conventional methods for detecting mutations in large fragments of TSC genes are Southern hybridization and in situ fluorescence hybridization, but these methods have the disadvantages of requiring large amounts of DNA. Using long-fragment PCR requires only a small amount of genomic DNA, and it is easy to confirm the sequence of the mutation site.