PCR has two limitations:
a. short sizes of amplified products (<5 kb). This is solved by doing Long-range PCR (up to tens of Kb long) which uses a mixture of two heat stable polymerases that provide optimal levels of DNA synthesis as well as a 3’ -> 5’ exonuclease activity.
b. low yields of amplifications which is resolved by cloning the PCR amplified DNA fragment in a vector then propagating the vector in a cell based system (clone by A/T cloning or by using anchored PCR primers).
General Applications of PCR:
PCR has 3 major advantages:
- rapid
- sensitive
- robust (possible to amplify DNA from damaged tissues or degraded DNA)
Primer specificity is very important in PCR. Several modifications have been developed to reduce nonspecific binding (see Box 5.1):
- Hot-start PCR
- Nested PCR
- Touch-down PCR
The correct base pairing at the extreme 3’ end of bound primers is a requirement for producing a PCR product. This allowed the use of PCR to distinguish between alleles of the same gene that differ in a single nucleotide (allele-specific PCR). This method is known as ARMS (amplification refractory mutation system). 
Degenerate oligonucleotide primed PCR (DOP-PCR) allow the amplification of a different but closely related genes (novel genes) at the same time.
Indiscriminate amplification of whole genomes can be performed using linker-primed PCR (ligation adaptor PCR).
PCR could be used to amplify unknown DNA sequences neighboring a known sequence. Such methods include anchored PCR, inverse PCR, RACE-PCR.
