PCR hot start using primers with the structure of molecular beacons (hairpin-like structure)

Abstract

A new technique of PCR hot start using oligonucleotide primers with a stem-loop structure is developed here. The molecular beacon oligonucleotide structure without any chromophore addition to the ends was used. The 3'-end sequence of the primers was complementary to the target and five or six nucleotides complementary to the 3'-end were added to the 5'-end. During preparation of the reaction mixture and initial heating, the oligonucleotide has a stem-loop structure and cannot serve as an effective primer for DNA polymerase. After heating to the annealing temperature it acquires a linear structure and primer extension can begin.

Figure 1

Effect of the molecular beacon primer on PCR specificity and sensitivity. (A) The target was M.tuberculosis DNA. All reaction mixtures contained 0.5 µg of human placental DNA. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder), the prominent band is at 500 bp; lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, ∼1000 genome copies; lanes 3, 6, 9 and 12, 100 copies; lanes 4, 7, 10 and 13, 10 copies; lanes 2–7, platinum Taq DNA polymerase; lanes 8–13, normal Taq DNA polymerase. (B) Detection of M.tuberculosis DNA in clinical sample by the described technique. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder); lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, DNA sample from sputum; lanes 3, 6, 9 and 12, ∼1000 genome copies; lanes 4, 7, 10 and 13, ∼100 genome copies; lanes 2–7, platinum Taq DNA polymerase; lanes 8–13, normal Taq DNA polymerase.

Figure 1

Effect of the molecular beacon primer on PCR specificity and sensitivity. (A) The target was M.tuberculosis DNA. All reaction mixtures contained 0.5 µg of human placental DNA. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder), the prominent band is at 500 bp; lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, ∼1000 genome copies; lanes 3, 6, 9 and 12, 100 copies; lanes 4, 7, 10 and 13, 10 copies; lanes 2–7, platinum Taq DNA polymerase; lanes 8–13, normal Taq DNA polymerase. (B) Detection of M.tuberculosis DNA in clinical sample by the described technique. Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder); lanes 2–4 and 8–10, reverse primer as molecular beacon; lanes 5–7 and 11–13, linear reverse primer; lanes 2, 5, 8 and 11, DNA sample from sputum; lanes 3, 6, 9 and 12, ∼1000 genome copies; lanes 4, 7, 10 and 13, ∼100 genome copies; lanes 2–7, platinum Taq DNA polymerase; lanes 8–13, normal Taq DNA polymerase.

Figure 2

Effect of the molecular beacon primer on the PCR specificity and sensitivity. The target was human DNA (exon 4 of p53 gene). Gel electrophoresis in 2% agarose of the amplification products. Lane 1, DNA size marker (100 bp ladder), the prominent band is 500 bp; lanes 2, 3 and 6, 7, reverse primer as molecular beacon; lanes 4, 5 and 8, 9, linear reverse primer; lanes 2, 4, 6 and 8, ∼1000 genome copies; lanes 3, 5, 7 and 9, ∼100 genome copies; lanes 2–5, platinum Taq DNA polymerase; lanes 8 and 9, usual Taq DNA polymerase. The platinum Taq DNA polymerase gave good amplification from 100 000+ genome copies (not shown).