RESUMEN
The reported genome-walking methods still suffer from some deficiencies, such as cumbersome experimental steps, short target amplicon, or deep background. Here, a simple and practical fork PCR was proposed for genome-walking. The fork PCR employs a fork primer set of three random oligomers to implement walking task. In primary fork PCR, the low-stringency amplification cycle mediates the random binding of primary fork primer to some places on genome, producing a batch of single-stranded DNAs. In the subsequent high-stringency amplification, the target single-strand is processed into double-strand by the site-specific primer, but a non-target single-stranded DNA cannot be processed by any primer. As a result, only the target DNA can be exponentially amplified in the remaining high-stringency cycles. Secondary/tertiary nested fork PCR(s) further magnifies the amplification difference between the both DNAs by selectively enriching target DNA. The applicability of fork PCR was validated by walking several gene loci. The fork PCR could be a perspective substitution for the existing genome-walking schemes.
RESUMEN
In order to understand the research status of HUANG Fu-mi's A-B Classic of Acupuncture and Moxibustion, 83 pieces of literature that have been publically published since 1978 were analyzed. A summary was performed according to 7 aspects including version origin, emendation, arrangement and summary, theory discussion, clinical research, value evaluation, the rest questions, etc. It is believed that deep research work has been done on explanations, emendation and translation regarding A-B Classic of Acupuncture and Moxibustion. However, the exploration work on the theory and application is far from enough. Therefore, beginning from historical and cultural background, we should analyze the humanistic spirit of HUANG Fu-mi combined with A-B Classic of Acupuncture and Moxibustion to perform a deep exploration, so we could have a thorough and comprehensive research on HUANG Fu-mi's academic thoughts.