Simple innovative adaptor to improve genome walking with convenient PCR

Background Various polymerase chain reaction (PCR)-based methods have been applied for the development of genome walking (GW) technique. These methods which could be based on the application of restriction enzymes or primers have various efficiencies to identify the unknown nucleotide sequences. The present study was conducted to design a new innovative double-strand adaptor using MAP30 gene sequence of Momordica charantia plant as a model to improve genome walking with convenient PCR. Results The adaptor was designed using multiple restriction sites of Hind III, BamH I, EcoR I, and Bgl II enzymes with no restriction site in a known sequence of the MAP30 gene. In addition, no modification was required to add phosphate, amine, or other groups to the adaptor, since restriction enzyme digestion of double-strand adaptor provided the 5′ phosphate group. Here, preparation of the phosphate group in the genomic DNA of the plant digestion with restriction enzymes was performed followed by ligation with digested adaptor containing 5′ phosphate group. Conclusion PCR was done to amplify the unknown sequence using MAP30 gene-specific primer and adaptor primer. Results confirmed the ability of the technique for successful identification of the sequence. Consequently, a newly designed adaptor in the developed technique reduced the time and cost of the method compared to the conventional genome walking; also, cloning and culturing of bacterial steps could be eliminated.

Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking

Genome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3′-ends. Upstream of the palindromes there is a degenerate sequence (8–12 nucleotides long); defined adapters are present at the 5′-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.

Virtual Genome Walking: Generating gene models for the salamander Ambystoma mexicanum

Large repeat rich genomes present challenges for assembly and identification of gene models with short read technologies. Here we present a method we call Virtual Genome Walking which uses an iterative assembly approach to first identify exons from de-novo assembled transcripts and assemble whole genome reads against each exon. This process is iterated allowing the extension of exons. These linked assemblies are refined to generate gene models including upstream and downstream genomic sequence as well as intronic sequence. We test this method using a 20X genomic read set for the axolotl, the genome of which is estimated to be 30 Gb in size. These reads were previously reported to be effectively impossible to assemble. Here we provide almost 1 Gb of assembled sequence describing over 19,000 gene models for the axolotl. Gene models stop assembling either due to localised low coverage in the genomic reads, or the presence of repeats. We validate our observations by comparison with previously published axolotl bacterial artificial chromosome (BAC) sequences. In addition we analysed axolotl intron length, intron-exon structure, repeat content and synteny. These gene-models, sequences and annotations are freely available for download from https://tinyurl.com/y8gydc6n. The software pipeline including a docker image is available from https://github.com/LooseLab/iterassemble. These methods will increase the value of low coverage sequencing of understudied model systems.

Identifikasi dan isolasi promoter gen pembungaan kakao TcLFY Identification and isolation for promoter of TcLFY cacao flowering gene

SummaryPromoter is a regulator of geneexpression for a phenotype or trait carried bythe gene. In the structure, a promoter locatedbeyond the 5’ end of the open reading frame ofthe gene on which its expression is regulated.This research aimed to isolate the DNAfragment flanking TcLFY at the 5’ end and toanalyze whether the fragment has charac-teristics of the promoter, primarily the coremotifs of promoter. Using Genome Walkingtechnique, DNA fragments flanking the TcLFYgene at its 5’ end was isolated. Analysis of theDNA sequence was done using onlinecomputer software accessible through web sitewww.softberry.com and an entry sequence ofthe flanking DNA fragment along with the 2.5kb TcLFY sequence. The result indicated thatthe flanking fragment has core motifs for apromoter at proper positions, which are TATAbox at position –80, CAT boxes (CCAAT) at -387 and –626, and GC boxes that are known asUAS were found at the -323 and –537positions. To obtain a conclusive result, thispromoter sequence needs to be furtherexamined to confirm its function.RingkasanPromoter merupakan pengendali ekspresigen untuk memunculkan fenotipe atau karakteryang dibawa oleh gen tersebut. Di dalamstrukturnya, promoter umumnya terletak didaerah ujung 5’ gen yang dikendalikanekspresinya. Tujuan penelitian ini adalahmendapatkan fragmen DNA yang mengapitgen pengendali pembungaan kakao (TcLFY)dan menganalisisnya apakah memilikikarakteristik promoter, yaitu mengandungmotif-motif inti (core motifs) dari promoter.Dengan teknik Genome Walking, fragmenDNA pengapit gen TcLFY di ujung 5’ dapatdiisolasi. Analisis sekuen menggunakanperangkat lunak komputer online (www.softberry.com) dengan input data fragmentersebut ditambah gen TcLFY 2,5 kb dibawahnya, mengindikasikan adanya beberapamotif inti promoter pada posisi yang sesuai,yaitu kotak TATA pada lokasi –80, kotak CAT(CCAAT) di posisi -387 dan –626, dan kotakGC yang merupakan UAS dijumpai padalokasi -323 dan –537. Untuk memperoleh hasilyang bersifat konklusif, sekuen promoter inimasih perlu diuji fungsinya.

Identifikasi dan isolasi promoter gen pembungaan kakao TcLFY Identification and isolation for promoter of TcLFY cacao flowering gene

SummaryPromoter is a regulator of geneexpression for a phenotype or trait carried bythe gene. In the structure, a promoter locatedbeyond the 5’ end of the open reading frame ofthe gene on which its expression is regulated.This research aimed to isolate the DNAfragment flanking TcLFY at the 5’ end and toanalyze whether the fragment has charac-teristics of the promoter, primarily the coremotifs of promoter. Using Genome Walkingtechnique, DNA fragments flanking the TcLFYgene at its 5’ end was isolated. Analysis of theDNA sequence was done using onlinecomputer software accessible through web sitewww.softberry.com and an entry sequence ofthe flanking DNA fragment along with the 2.5kb TcLFY sequence. The result indicated thatthe flanking fragment has core motifs for apromoter at proper positions, which are TATAbox at position –80, CAT boxes (CCAAT) at -387 and –626, and GC boxes that are known asUAS were found at the -323 and –537positions. To obtain a conclusive result, thispromoter sequence needs to be furtherexamined to confirm its function.RingkasanPromoter merupakan pengendali ekspresigen untuk memunculkan fenotipe atau karakteryang dibawa oleh gen tersebut. Di dalamstrukturnya, promoter umumnya terletak didaerah ujung 5’ gen yang dikendalikanekspresinya. Tujuan penelitian ini adalahmendapatkan fragmen DNA yang mengapitgen pengendali pembungaan kakao (TcLFY)dan menganalisisnya apakah memilikikarakteristik promoter, yaitu mengandungmotif-motif inti (core motifs) dari promoter.Dengan teknik Genome Walking, fragmenDNA pengapit gen TcLFY di ujung 5’ dapatdiisolasi. Analisis sekuen menggunakanperangkat lunak komputer online (www.softberry.com) dengan input data fragmentersebut ditambah gen TcLFY 2,5 kb dibawahnya, mengindikasikan adanya beberapamotif inti promoter pada posisi yang sesuai,yaitu kotak TATA pada lokasi –80, kotak CAT(CCAAT) di posisi -387 dan –626, dan kotakGC yang merupakan UAS dijumpai padalokasi -323 dan –537. Untuk memperoleh hasilyang bersifat konklusif, sekuen promoter inimasih perlu diuji fungsinya.