![]() It has typically been most useful for molecular systematics at the species level, and even within species (e.g., to identify geographic races). Notes on ITS: The ITS region is the most widely sequenced DNA region for Fungi. ITS (internal transcribed spacer) region primers Most of the data in our Agaricales LSU database consists of the first 900 bases from the LSU gene (we typically amplify using primers 5.8SR + LR7, followed by sequencing using primers LR5, LR16, LR0R, and LR3R). Most molecular systematics studies only utilize the first 600-900 bases from the LSU gene, which includes three divergent domains (D1, D2, D3) that are among the most variable regions within the entire gene (much of the LSU is invariant even across widely divergent taxa). Note: Many of these primers were first reported in several papers (Hopple and Vilgalys, Hibbett and Vilgalys, Vilgalys and Hester, ). NS = primers described by White et al., 1990 SSU (small-subunit 18S RNA) primer sequencesīMB = “universal” SSU primers developed by Lane et al., 1985 ![]() Another useful source of primer information may be found in Gargas & Depriest (1996) and at the Tom Bruns lab web site. See Gerbi (1986) for a general introduction to the molecular biology and evolution of rDNA in other eukaryotes. Primers 5.8SR and LR7 include these restriction sites, which makes them convenient for cloning.įor those who aren’t familiar with rDNA and fungal systematics, several excellent reviews are available on fungi (Hibbett, 1992) and generally for eukaryotes (Hillis and Dixon, 1991). Nearly all basidiomycetes we’ve studied share an EcoRI site within the 5.8S RNA gene along with a BglII site halfway into the LSU RNA sequence. Several restriction sites for EcoRI and BglII are conserved in the rDNA of fungi. In some groups (mostly basidiomcyetes and some ascomycetous yeasts), each repeat also has a separately transcribed coding region for 5S RNA whose position and direction of transcription may vay among groups. Each repeat unit has coding regions for one major transcript (containing the primary rRNAs for a single ribosome), punctuated by one or more intergenic spacer (IGS) regions. The nuclear-encoded ribosomal RNA genes (rDNA) of fungi exist as a multiple-copy gene family comprised of highly similar DNA sequences (typically from 8-12 kb each) arranged in a head-to-toe manner. Multiple copies of near-identical rDNA operons are organized in a head-to-toe manner on a single chromosome. Organization of ribosomal RNA genes in fungi. This information is provided freely and may be passed on to anyone who wants to use it. All other primers are complementary to the coding strand. Primers ending with “R” represent the coding strand (same as RNA). Standard symbols are used for the four primary nucleotides variable positions are indicated as follows: P=A,G / Q=C,T / R=A,T / V=A,C / W=G,T. Together, these primers span most of the nuclear rDNA coding region (see figures), permitting amplification of any desired region. All of these primers were first identified and tested by our own lab based on consensus between the published large and small subunit RNA sequences from fungi, plants and other eukaryotes sources of other useful primer sequences from published literature are also indicated. Over the years, our lab has compiled a useful list of conserved primer sequences useful for amplification and sequencing of nuclear rDNA from most major groups of fungi (primarily Eumycota), as well as other eukaryotes. ![]() Primer sites within the ribosomal RNA genes of fungi
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |