False Positives Caused by Single Primer in DDRT Analysis of Soybean

[Objective] The aim was to explore the reasons of false positives in Different Display Reverse Transcription（DDRT）analysis.[Method] Soybean varieties ＂Jilin 30＂ and ＂Tongnong 13＂ were used as materials to carry out analysis on false positives in DDRT analysis.[Result] An important origin of false positives appeared in DDRT analysis was the non-specific amplification caused by the combination of single primer and cDNA.The parallel PCR test of single primer should be set so as to verify whether the obtained fragments were the false positives or the PCR productions combined with single primer.[Conclusion] This study had provided basis for improving the success rate of DDRT experiment.

Molecular Marker Techniques Using Single Primers and Their Advances

Molecular marker techniques have been widely applied in the fields of genetic diversity analysis,germplasm resources identification,molecular fingerprint and genetic linkage map construction,QTL mapping and molecular assisted breeding.On the basis of stating the concept of molecular marker techniques based on single primer amplification reactions,this study focused on the sorting and induction of single-primer molecular marker techniques,and expounded their derivative development.Finally,the application prospect and future expectation of single-primer molecular marker techniques were described in detail.The purpose of this study was to clarify the types of molecular marker techniques based on single primer amplification reactions,so that researchers can quickly and conveniently select molecular marker techniques according to their own specific scientific research conditions.

An easy PCR-based genome-walking method for getting the unknown 5’flanking region of a Scenedesmus sp.

Objective:To develop the current single primer PCR-based genome-walking method with Scenedesmus sp.Methods:The unknown 5’and/or 3’flanking regions for a specific conserved sequence were optimized and the current single primer PCR-based genome-walking method were developed.Alignment was between the related species of microalga and Scenedesmus sp.For 18S rDNA,we selected the species Scenedesmus sp.,Chlorella sp.,and Chlamydomonas sp.For the rbcL gene from the chloroplast genome,alignment was done between Scenedesmus sp.,and Chlamydomonas sp.Results:Obtaining a small conserved sequence for any gene family is something that can be achieved quite easily.However,identifying the whole gene is often difficult.After investigating and testing,some of the current protocols using to get the unknown 5’and/or 3’flanking regions for a specific conserved sequence,we developed the current single primer PCR-based genome-walking method.We performed two consecutive PCR reactions;band extraction and the PCR product were sequenced.We got our results by testing the method on three genes from the total DNA of Scenedesmus sp.;two genes had a fully known sequence in gene bank(18S rDNA and rbcL),but the third one has not yet been identified(rbcS).We designed our primers based on the alignment between the related species and to each other.We also tested two different DNA polymerases Ex Taq and TLA polymerase.Conclusions:Results from our study suggest that Ex Taq is the most suitable polymerase for the current protocol.