Primer Extension Reaction Assays for Incorporation of Deoxynucleotide Analogue into DNA

Incorporation of deoxynucleotide analogues into DNA is important for the expansion of DNA functions.Primer extension reactions are commonly used for the assay of such reaction events.However,current assay protocols generally rely on radiolabeling,fluorescence reporter labeling,or removal of specific deoxynucleotide triphosphate in the reaction mixture.Herein we report on the design of two novel assay protocols that utilize a dideoxynucleo-tide-terminated template strand and a phosphorothiolate-modified deoxynucleotide-terminated template strand.We designed and synthesized a deoxyuridine triphosphate analogue(dU^(*)TP)containing 2-bromoisobutyryl group and demonstrated that it could be well recognized byφ29DNA polymerase,E.coli DNA polymerase I Klenow Fragment,Bst DNA polymerase Large Fragment,and E.coli DNA polymerase I Klenow Fragment(exo^(−)),which translated to effective incorporation of dU^(*)TP into DNA.dU^(*)TP was also successfully incorporated into extremely long single-stranded DNA at high-density usingφ29 DNA polymerase by rolling circle amplification.

DNA sequencing by synthesis with degenerate primers

The degenerate primer-based sequencing was developed by a synthesis method （DP-SBS） for high-throughput DNA sequencing, in which a set of degenerate primers are hybridized on the arrayed DNA templates and extended by DNA polymerase on microarrays. In this method, a different set of degenerate primers containing a given number （n） of degenerate nucleotides at the 3＇-ends were annealed to the sequenced templates that were immobilized on the solid surface. The nucleotides （n＋1） on the template sequences were determined by detecting the incorporation of fluorescent labeled nucleotides. The fluorescent labeled nucleotide was incorporated into the primer in a base-specific manner after the enzymatic primer extension reactions and nine-base length were read out accurately. The main advantage of the DP-SBS is that the method only uses very conventional biochemical reagents and avoids the complicated special chemical reagents for removing the labeled nucleotides and reactivating the primer for further extension. From the present study, it is found that the DP-SBS method is reliable, simple, and cost-effective for laboratory-sequencing a large amount of short DNA fragments.

Gene Analysis of Free Fetal DNA in Maternal Plasma

To investigate the feasibility of using free fetal DNA from maternal plasma as the source of fetal material in non invasive prenatal diagnosis, SRY gene of free DNA in maternal blood of 65 samples were analyzed by using primer extension preamplication (PEP) and probe microplate hybridization techniques. The results showed that the detection rate of SRY gene in maternal blood from women carrying male fetuses detected by probe microplate hybridization alone and probe microplate hybridization with PEP were 76.09 % (35/46) and 95.65 % (44/46) respectively, and there was a significant difference between them. The non detection rate of SRY gene in blood samples from women carrying female fetus was 100 % (19/19). It is indicated that probe microplate hybridization was an effective method in detecting trace fetal DNA from maternal plasma and the sensitivity could be substantially improved by combined use of the two techniques. Analysis of fetal DNA in maternal plasma can serve as an alternative for non invasive prenatal diagnosis.

Evolutionary divergence in Chenopodium and validation of SNPs in chloroplast rbcL and matK genes by allele-specific PCR for development of Chenopodium quinoa-specific markers

The genus Chenopodium comprises about 150 species, of which Chenopodium quinoa and C. album are important for their nutritional value. Evaluation of variation in qualitative morphological traits of plants and SNPs in chloroplast rbc L and mat K gene sequences in 19 accessions representing C. quinoa and C. album indicated that the accessions IC-411824 and IC-411825,which have white seeds, belong to C. quinoa rather than C. album. This observation was also supported by a time tree that indicated IC-411824 and IC-411825 to be a sister clade to accessions of C. quinoa with an estimated age of 1.2 Mya. Whereas multiple alignments of rbc L gene sequences from the 19 accessions revealed 1.26% parsimony-informative sites with 0.68%interspecific sequence diversity, alignment of nucleotide sequences of amplicons representing the mat K gene revealed 4.97% parsimony-informative sites and 2.81% interspecific sequence diversity. Validation of SNPs in the cp rbc L and mat K regions of 36 accessions belonging to C. quinoa and C. album was performed by allele-specific PCR with primers carrying a single base change at the 3′ end. We report the first C. quinoa-specific SNP-based primer, R1RQ-AFR,designed from rbc L sequences, that could differentiate quinoa from 64 genera including13 species of the genus Chenopodium. With an estimated age of 10.5–4.1 million years(Myr), the Himalayan chenopods are evolutionarily younger than the Andean chenopods. The results establish the paraphyletic origin of the genus Chenopodium.

Noninvasive Prenatal Diagnosis of Fetal Sex by Single-cell PEP-PCR Method

A new method for noninvasive prenatal diagnosis of fetal sex was developed by using single cell PEP PCR techniques. Micromamipulation techniques were used to obtain single fetal cells from 273 maternal blood samples. The genome of single cells was preamplified by PEP and SRY genes were analyzed by PCR method. The SRY genes of 149 samples were detected by the new method among 153 samples carrying male fetus, while 119 out of 120 samples carrying female fetus were proved negative for SRY genes. The sensitivity and specificity of the new method were 97.39 % and 99.17 % respectively and the correct rate was 98.17 %. The new method has the advantage of high sensitivity and specificity in noninvasive prenatal diagnosis of fetal sex and provides the basis of other researches such as sex linked inherited diseases.

Single Cell Analysis of Dystrophin and SRY Gene by Using Whole Genome Amplification

ve To develop a reliable and sensitive method for detection of sex and multi-loci of Duchenne muscular dystrophy (DMD) gene in single cell

Analysis of the 5' flanking sequence of the human norepinephrine transporter gene

The human norepinephrine transporter(NET) gene was cloned and structurally analyzed. The far 5’ fragment containing exon 1 (a non-coding exon) and exon 2 was sequenced. The transcription start site of the gene in human brain stem tissue was determined by primer extension analysis. It was found that the gene could be transcribed from multiple starting points. The 5’ flanking sequence contains a proximal G-C rich region, one possible GSG elemeflt and several SP1 sites. However it does not contain TATA box and CAAT box motifS. Gel shift analysis with nuclear extracts from different tissues of mouse shows that the G-C rich region may be involved in tissue specific expression of the gene.

Review:Whole genome amplification in preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.

The History and Advances of Reversible Terminators Used in New Generations of Sequencing Technology

DNA sequencing using reversible terminators, as one sequencing by synthesis strategy, has garnered a great deal of interest due to its popular application in the second-generation high-throughput DNA sequencing technology. In this review, we provided its history of develop- ment, classification, and working mechanism of this technology. We also outlined the screening strategies for DNA polymerases to accommodate the reversible terminators as substrates during polymerization; particularly, we introduced the ＂REAP＂ method developed by us. At the end of this review, we discussed current limitations of this approach and provided potential solutions to extend its application.