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Using the website

Starting with a sequence   A DNA sequence can be BLASTed at the BLAST page. Ds insertions within and near each match are returned. While it is not necessary to give a title to a single sequence, multiple sequences pasted into the text area must each be preceded by a line containing a ">" character, a title, and a paragraph return character (i.e. FASTA format). Protein sequences can also be BLASTed with the TBLASTN program. More regions of the genome having homology to the query can be searched by increasing the expect value to allow less exact matches.

Starting with a GenBank accession   Accessions can be searched on the Annotations page. Ds insertions within and near the accession are returned. Results can be limited by the location of the insertion using the menu below the text box.

Starting with keywords or an annotation   The best strategy is to obtain the annotation for a gene of interest from GenBank then enter words from the annotation in various combinations on the Annotations page. Specificity can be obtained by grouping the words into phrases, however where a more general search is desired queries should be kept short. The search is case-insensitive.

Annotations

How are annotations generated?   Sequences flanking Ds insertions are sent by an automated program for BLAST search against the TIGR chromosome assemblies and against all sequences in GenBank, generating the two annotations associated with each sequence. From the TIGR annotation of the genome, the chromosome, nucleotide position, published name, common name, locus, feature name, and public comment are obtained from the nearest feature to the match. By noting the primer used to generate the sequence, the orientation of the Ds element relative to the annotated feature is determined. Likewise, BLAST results from GenBank are parsed for GI, accession, gene, product, note, and evidence. Experimental matches (genes, mRNAs, CDSs) are taken in preference to theoretical annotations. Thus, new experimental data can be incorporated into the information in genetrap annotations.

What fields are searched?   The fields searched by the keyword search program are feature name, locus, published name, common name, public comment from TIGR, and gene, product, note from GenBank. These encompass BAC names, ESTs, enzyme or protein names, and gene names.

How can searches be optimized?   Wild cards (the '*' character) can be used to represent any number of additional characters for widening the scope of a search. The search is not case sensitive except in the case of operators: the terms "AND", "OR", and "NOT" must be capitalized. To limit the number of spurious or uninteresting matches returned by a search use the AND or NOT keywords to add terms that must or must not be present.

Why do annotations for some lines disagree?   Sequences are generated by Thermal Asymmetric Interlaced PCR. While this protocol generally produces reliable sequence, it can occasionally generate artifacts from elewhere in the genome. Researchers are advised that the sequences and annotations contained in the database are experimental and should be confirmed by specific PCR reactions between a gene-specific primer and a Ds-specific primer. back to top

Phenotypes

How are phenotypes scored?   Phenotypes such as semi-sterility are scored after self-fertilization of the F2. Other phenotypes are recorded as the lines are propagated.

Do lines with no listed phenotype really have no phenotype?   The simple answer: No. Subtle phenotypes may not be recorded and many lines have not been extensively screened past the F2 generation. back to top

BLAST

How is the BLAST search done?   Sequences submitted as queries to the genetrap website are BLASTed against the chromosome assemblies of TIGR and their absolute positions on the chromosomes are noted. The sequences flanking Ds insertions are also BLASTed against these same chromosome sequences. Thus, once the coordinates of the query are known, Ds insertions whose coordinates fall within or near the query are retrieved and diplayed. Currently TIGR's assembly version from Jan 2002 is used.

How can I get sequences to do my own BLAST analysis?   Flanking sequences for each line can be obtained by clicking the link above each sequence in the profile of a line. It is then a simple task to select the sequence, copy it, and paste it into the query box of any website performing BLAST searches.

How are the sequences generated?   Sequences flanking Ds insertions are generated by Thermal Asymmetric Interlaced PCR (TAIL PCR). The technique uses nested Ds-specific primers on the transposon side and an arbitrary degenerate primer to prime on the genome side. Successful amplification is seen in about 70% of reactions therefore some lines will not have sequences from both the 3' and 5' ends of the Ds element. The sequences of the primers we use are:

 Ds3-1 ACC CGA CCG GAT CGT ATC GGT
Ds3-2 CGA TTA CCG TAT TTA TCC CGT TC
Ds3-4 CCG TCC CGC AAG TTA AAT ATG
Ds5-1 GAA ACG GTC GGG AAA CTA GCT CTA C
Ds5-2 CCG TTT TGT ATA TCC CGT TTC CGT
Ds5-4 TAC GAT AAC GGT CGG TAC GG
AD2 NGT CGA SWG ANA WGA A

Sequences generated by the Ds5 primers are oriented in the same direction as the GUS gene in the Ds element. Sequences generated with the Ds3 primers are opposite.

Why do sequences for some lines disagree?   TAIL PCR can sometimes produce artifactual products from elsewhere in the genome. When sequences from a line disagree in genomic position, and as a general practice, researchers are advised to confirm the position of the Ds element with specifically designed primers. back to top

Expression

How is expression assayed?   Seedlings and, lately, flowers are stained for beta-glucuronidase (GUS) activity in the following buffer:

  1 mM X-gluc
100 mM Na phosphate pH 7
10 mM EDTA
0.1% Triton X100
100 mg/l chloramphenicol
optional: 2 mM ferricyanide

Obtaining and working with genetrap lines

How can I obtain a genetrap line?   Send an email message to Joe Simorowski (simorows@cshl.edu). Do not send a completed MTA at this stage since some of the lines may not be available. After receiving a reply, complete the MTA and send it along with applicable payment to:

  Joe Simorowski
Cold Spring Harbor Laboratory
1 Bungtown Road
Cold Spring Harbor, NY
USA 11724

What should I do when I receive a line?   Confirm the site of the Ds insertion by designing a gene-specific primer to work with one of the Ds specific primers listed above. The segregation of the kanamycin resistance marker should also be observed on 50 mg/l kanamycin. Every effort is made to ensure that the lines each have one Ds element and to obtain accurate flanking sequence, however researchers are urged to confirm that this is the case.

How can I put information in the database?   Contact Bruce May (may@cshl.edu) with any data you would like to contribute to the database. Corrections of data already in the database are especially appreciated. back to top