Biology 382 - Project 1 Information

Genes to select from:
Bio 382 - dhs / sdr / akr / sdh / adh genes to clone for 2010:

gene name  LG  temporary gene name
dhs-5	   II	-
dhs-27	    X	-
dhs-13	    V	-
F53C11.3    V  	usr-A
T03F6.1	   III  usr-B
F02C12.2    X	usr-C
C06E4.6	   IV	usr-D
R05D8.9	   V	usr-E
C06E4.6	   IV	usr-F
C01G12.5   II	usr-G
F26D2.15    V 	usr-H
C06E4.3	   IV	usr-J
R05D8.7	   V	usr-K

*usr - unknown specificity reductase
Each gene encodes a possible substitute for the SR in BH4 synthesis in C. elegans.

  • BH4 Information for the research project(s)
  • Directory with project documents & images

    Biology 382 - Project 1 Tasks

    1. Select two genes to work with
    2. Via Wormbase, find the sequence of your gene-of-interest (GOI)
    3. Identify introns & exons (current gene model)
    4. Identify exon regions fully confirmed by cDNAs
    5. Design primers that include coding sequence (aim, if possible, for a minimum of about 500 bp of coding sequence, with amplicon of 1000 bp or less - could be entirely coding sequence if such an exon is available).
      Primer3 for primer design

    6. Test primers via e-PCR
      Reverse e-PCR

      Put selected primers into window, select C. elegans genome database
      Put in size found in Primer3 design, with 1000 bp deviation
      Select Primer Alignment Quality: 2 mismatches, 2 gaps

      You should get only the product predicted from your Primer3 analysis

    7. Test primers for self complementarity here: OligoCalc: Oligonucleotide Properties Calculator

    8. BLAST proposed amplified sequences (amplicons) to look for similarity in C.e. genome to avoid 'off-target' effects. Avoid regions longer than about 21 bps that have >80% identity. This could particularly be a problem with intronic sequence.

      NCBI BLAST Server

    9. Additional study of GOI's (need not be done immediately):
        a. Find orthologs in C.e. relatives, align protein sequences
        b. Do protein multiple sequence alignments of homologs from Ce, Dm, Hs, etc. (wider phylogenetic range to identify conserved regions)
        c. Literature searches and reading to learn about functions of the genes in other organisms.