Skip to main content
Article thumbnail
Location of Repository

Revised Selection Criteria for Candidate Restriction Enzymes in Genome Walking

By Ali Taheri, Stephen J. Robinson, Isobel Parkin and Margaret Y. Gruber

Abstract

A new method to improve the efficiency of flanking sequence identification by genome walking was developed based on an expanded, sequential list of criteria for selecting candidate enzymes, plus several other optimization steps. These criteria include: step (1) initially choosing the most appropriate restriction enzyme according to the average fragment size produced by each enzyme determined using in silico digestion of genomic DNA, step (2) evaluating the in silico frequency of fragment size distribution between individual chromosomes, step (3) selecting those enzymes that generate fragments with the majority between 100 bp and 3,000 bp, step (4) weighing the advantages and disadvantages of blunt-end sites vs. cohesive-end sites, step (5) elimination of methylation sensitive enzymes with methylation-insensitive isoschizomers, and step (6) elimination of enzymes with recognition sites within the binary vector sequence (T-DNA and plasmid backbone). Step (7) includes the selection of a second restriction enzyme with highest number of recognition sites within regions not covered by the first restriction enzyme. Step (8) considers primer and adapter sequence optimization, selecting the best adapter-primer pairs according to their hairpin/dimers and secondary structure. In step (9), the efficiency of genomic library development was improved by column-filtration of digested DNA to remove restriction enzyme and phosphatase enzyme, and most important, to remove small genomic fragments (<100 bp) lacking the T-DNA insertion, hence improving the chance of ligation between adapters and fragments harbouring a T-DNA. Two enzymes, NsiI and NdeI, fit these criteria for the Arabidopsis thaliana genome. Their efficiency was assessed using 54 T3 lines from an Arabidopsis SK enhancer population. Over 70% success rate was achieved in amplifying the flanking sequences of these lines. This strategy was also tested with Brachypodium distachyon to demonstrate its applicability to other larger genomes

Topics: Research Article
Publisher: Public Library of Science
OAI identifier: oai:pubmedcentral.nih.gov:3324424
Provided by: PubMed Central
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • http://www.pubmedcentral.nih.g... (external link)
  • Suggested articles

    Citations

    1. (2002). A new technique to prevent self-ligation of DNA.
    2. (2009). A protocol for efficiently retrieving and characterizing flanking sequence tags (FSTs) in Brachypodium distachyon T-DNA insertional mutants.
    3. (2000). Activation Tagging in Arabidopsis.
    4. (1998). Adaptor Ligation-Based Polymerase Chain Reaction-Mediated Walking.
    5. (2009). An archived activation tagged population of Arabidopsis thaliana to facilitate forward genetics approaches.
    6. (2007). An improved method to identify the TDNA insertion site in transgenic Arabidopsis thaliana genome.
    7. (1995). An improved PCR method for walking in uncloned genomic DNA.
    8. (1986). An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends.
    9. (2000). Bleasby A
    10. (2002). Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis. OMICS:
    11. (1998). Cloning genes of Arabidopsis thaliana by chromosome walking.
    12. (1987). Detection in situ of genomic regulatory elements in Drosophila.
    13. (1995). Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.
    14. (2010). Gene cloning and DNA analysis : an introduction. Oxford ; Hoboken:
    15. (1989). Genome walking by single-specific-primer polymerase chain reaction:
    16. (2011). Genome-wide genetic marker discovery and genotyping using next-generation sequencing.
    17. (1996). High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR.
    18. (2004). High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics.
    19. (1990). Isolation of plant DNA from fresh tissue.
    20. (1996). Microbial Genetics: Jones and Bartlett Publishers (Boston).
    21. (1989). Molecular cloning: a laboratory manual. Molecular cloning: a laboratory manual Ed. 2. xxxviii +
    22. (1999). Mouse testicular extracts process DNA doublestrand breaks efficiently by DNA end-to-end joining.
    23. (2007). OligoCalc: an online oligonucleotide properties calculator.
    24. (1994). On Defining T-DNA.
    25. (2010). Restriction Site Extension PCR: A Novel Method for HighThroughput Characterization of Tagged DNA Fragments and Genome Walking.
    26. (1999). Screening of transgenic plants by amplification of unknown genomic DNA flanking T-DNA.
    27. (2009). Straight Walk: A modified method of ligation-mediated genome walking for plant species with large genomes.
    28. (2008). Substrate specificity of new methyl-directed DNA endonuclease GlaI.
    29. (2010). Template-blocking PCR: An advanced PCR technique for genome walking.
    30. (2008). Touchdown PCR for increased specificity and sensitivity in PCR amplification.
    31. (2008). Towards a better bowl of rice: assigning function to tens of thousands of rice genes.
    32. (1998). Uhle ´n M
    33. (1978). Use of the T4 Polynucleotide Ligase in The Joining of Flush-Ended DNA Segments Generated by Restriction Endonucleases.

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.