Haikuo Li

Single-base resolution and long-coverage sequencing based on single-molecule nanomanipulation

We show new approaches towards a novel single-molecule sequencing strategy which consists of high-resolution positioning isolation of overlapping DNA fragments with atomic force microscopy (AFM), subsequent single-molecule PCR amplification and conventional Sanger sequencing. In this study, a DNA labelling technique was used to guarantee the accuracy in positioning the target DNA. Single-molecule multiplex PCR was carried out to test the contamination. The results showed that the two overlapping DNA fragments isolated by AFM could be successfully sequenced with high quality and perfect contiguity, indicating that single-base resolution and long-coverage sequencing have been achieved simultaneously.

SIZE AND SURFACE EFFECT OF GOLD NANOPARTICLES (AuNPs) IN NANOGOLD-ASSISTED PCR

Recently, gold nanoparticles (AuNPs) were reported to increase the specificity and efficiency of the polymerase chain reaction (PCR). In this paper, we tested the enhancement of AuNPs with five different sizes on the specificity of two-round PCR. The results showed that, except 5.02 nm AuNPs, the AuNPs that could achieve the similar enhancement happened to have nearly the same total surface area. The surface effect seems to be the key factor of nanogold-assisted PCR.

Gold nanoparticle-assisted primer walking for closing the human chromosomal gap

The finished sequence of the human genome still contains 260 euchromatic gaps. All the PCR-based genome walking techniques used to close gaps have common limitations, such as low efficiency and low specificity. We herein describe a strategy to solve this problem by employing gold nanoparticles (AuNPs) to improve the efficiency in primer walking amplification. We used this strategy to close a gap in human chromosome 5 containing a DNA stretch composed of the 12SAT repeat. The obtained gap sequence is highly conserved among several mammalian genomes. The demonstrated AuNP-assisted primer walking strategy is capable of effectively improving the specificity of PCR amplification and enriching the yield of target DNA fragments; it offers a new avenue for closing gaps left by current sequencing methods.