ELXR References
Function
Input:   

EST or cDNA/mRNA fasta sequence or RefSeq accession number ELXR:

  1. Identifies the genomic contig containing your query checking in LocusLink annotation or via highest scoring BLAST sequence alignment to genomic nucleotide databases.
  2. Aligns query to genomic sequences using the Sim4 program. 
  3. Exon flanking PCR/sequencing primers are designed according to certain parameters using the Primer3 program

Blue Arrows represent forward and reverse primers for exon directed PCR and resquencing.  Blue rectangles indicate predicted exons from cDNA to genomic sequence alignments.

Output: sample

contains primer and alignment information as well as annotated genomic sequence to show exon locations (and start and stop codons if a RefSeq accession number is supplied as the query).

 
Applications

  • Scenario 1:  Design primers to amplify exons from genes in multiple individuals to detect sequence variations
  • Scenario 2:  Run multiple potential splice variant EST or cDNA sequences through ELXR to examine alternative splicing at the sequence level by comparing exon-annotated genomic sequences from ELXR output
  • Scenario 3:  Use the genomic flanking sequence option to control the amount of uptream sequence is returned in ELXR output to evaluate sequence features in gene promoters.
Program Control Hierarchy

Process Flow

Local ELXR Sequence Databases Utilized

Database Function Source
Human and mouse RefSeq Curated mRNA sequences retrieved by ELXR when a RefSeq accession number is used as input.  Currently only NM_XXXXXX type entries are supported NCBI
Human Genome Contigs Human BLAST vs mRNA queries to identify genomic contigs NCBI
High-Throughput Genomic Sequences (htg) Human & mouse secondary BLAST vs mRNA queries to identify genomic contigs NCBI
WGS Mouse Genome Supercontigs (MGSC) Mouse BLAST vs mRNA queries NCBI/Ensembl/MIT

ELXRdb Generation Parameters 11/1/02:

BLASTN
E value (E)  Threshold for acceptable genomic alignments =   1 e-40. 

The number of different alignments with scores equivalent to or better than S that are expected to occur in a database search by chance. The lower the E value, the more significant the score.

 
Remaining parameters are set to defaults
Sim4
N=1 High small external exon stringency

P=1 Remove Poly-A tails from mRNA sequence prior to alignment with genomic sequence

A=4 Output formatting
Remaining parameters are set to defaults
Primer3
No.to Return: 5 Max 3' Stability: 9.0
Max Mispriming: 12.0 Pair Max Mispriming: 24.0
Product Size  Min:   200         Opt:   350 Max:   450
Primer Size          Min:   18           Opt:   21               Max:   25
Primer Tm          Min:   30           Opt:   60               Max:   80
Primer GC%          Min:   30           Opt:   48               Max:   55
Max Self Complementarity: 8.0 Max 3' Self Complementarity: 3.0
Max #N's: 0 Max Poly-X: 5
CG Clamp: 0 Max Tm Difference: 2.0

Each primer is screened against repbase (Jurka, J. (2000). "RepBase Update: a database and an electronic journal of repetitive elements." Trends Genet. 9:418-420.) to screen out primer candidates which may promiscuously anneal to genomic templates.
ELXR Output Parameters
Sequencing buffer to between primer position and exon        =     30 bp
Output Sequence Flanking Genomic Slice Containing Gene  =  1000 bp
Output Sequence Flanking Each Aligned Exon                     =    300 bp