LMW glutenins represent approximately 40% of the total wheat gluten fraction. HMW glutenins represent a small family with a maximum of five of the six subunits expressed, while LMW glutenin genes are part of a much more complex gene family. HMW glutenins have been extensively studied because of their importance in dough rheology and their relative ease of analysis at the protein and DNA levels. Together, they are the major determinants of wheat bread-making quality. Wheat glutenins consist of high molecular weight glutenin subunits (HMW-GSs) and LMW-GSs. Wheat gluten is composed of two major groups of proteins, monomeric gliadins and polymeric glutenins. This method is very useful for rapid isolation and sequencing of gene families from BAC libraries. Here we describe a novel PCR method that combined hemi-nested PCR (a single internal primer) with touchdown PCR for primer-template mismatches. Touchdown (TD) PCR is a versatile one-step procedure for optimizing PCRs to obtain the specificity even if the degree of primer-template complementarity is not fully known. Nested PCR is useful in achieving the specificity by the amplification of an extended nucleotide sequence followed by the amplification of a region located within the first amplicon. In addition, they can not ensure specific amplification of individual genes from BAC clones containing two or more genes. These methods either require digestion and ligation procedures or are cumbersome, laborious and technically demanding. Several PCR methods are available for this purpose such as inverse PCR, ligation-mediated PCR, T-linker PCR and TAIL PCR. PCR amplification is a favorite strategy for isolation of unknown genomic sequences adjacent to known sequences. If a BAC clone contains two or more genes that display a high level of gene sequence identity such as the low molecular weight glutenin subunit (LMW-GS) genes, the primer walking using BAC DNA as template is likely not going to succeed due to multiple binding sites of the sequencing primer. If a BAC clone contains a single gene, complete gene sequence may be isolated through gene-specific PCR amplification and subsequent primer walking using BAC DNA as template and specific primers close to the end the known sequence obtained. They can also be used to isolate genes from complex gene families. It may be suitable for (i) isolation of other complex gene families and/or gene homologues from BAC libraries, (ii) for characterization of multi-copy repetitive elements pending availability of BAC libraries, and (iii) for filling in gaps in shotgun BAC sequencing.īacterial artificial chromosome (BAC) libraries are useful for genome analysis, comparative genomics, physical mapping and map-based cloning. This approach was fast, easy and cost-effective for isolation and sequencing of genes from complex gene families.

Comparison of two methods for purifying PCR products prior to sequencing showed that purification using MultiScreen 384-PCR filter plates had an advantage over ethanol purification because greater numbers of sequencing reactions could be performed from comparable volumes of PCR reactions. The hemi-nested TD PCR increased both specificity and yield by high and low annealing temperatures in two consecutive amplifications.

For the hemi-nested TD PCR, primers were designed based on the known sequences obtained and/or published. Two or more primer-template mismatches reduced PCR product yield approximately from 2-fold to 10-fold compared to PCR product yield without the primer-template mismatch. These mismatches included the transition mispairs G:T, T:G, A:C and the transversion mispairs A:A, A:G, G:G, G:A. Single or multiple mismatches were observed at 5' terminal, internal and the penultimate position, respectively. The effects of the universal primer-template mismatches on the efficiency of standard PCR amplification were investigated after assembly of sequences from different primers amplifying the same BAC clones. Resultsįor the primer-template mismatch PCR, the universal primers were designed based on conserved gene coding regions of consensus sequences. Here we report on an efficient approach for rapid isolation and sequencing of the low molecular weight glutenin subunit gene family from the 'Glenlea' wheat BAC library via primer-template mismatch PCR using universal primers, primer walking using hemi-nested touchdown (TD) PCR, and followed by direct sequencing of PCR products. Bacterial artificial chromosome (BAC) libraries can be useful for such work. Isolation of a large number of gene sequences from complex gene families with a high level of gene sequence identity from genomic DNA is therefore difficult and time-consuming. Hexaploid wheat ( Triticum aestivum L.) possesses a large genome that contains 1.6 × 10 10 bp of DNA.

Touchdown pcr vs nested pcr