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LAMA2 primers for mutation screening

(last modified November 29, 2005)

Contents

• DNA analysis
  • SSCA
• RNA analysis
  • PTT
  • RT-PCR+SSCA

DNA

SSCA

Legend:
Primer sequences for SSCA taken from Guicheney et al. [1998] - sequences were not checked. Exon: exon number according to these pages. Length: length of PCR fragment in bp. Forward primer /
reverse primer: sequence of forward and reverse primer. UPPERCASE = sequence in exon, * = sequence differs from that published (but agrees with cDNA Reference Sequence). PCR: touchdown-PCR with initial and final annealing temperature given. PCR was for 2 cycles at each temperature, going down in 1^oC steps and for 30 cycles at the final annealing temperature. Each cycle: 40 sec at 94^oC denaturation, 1 min extension at 72^oC, Final extension 2.5 min 72^oC.  NOTE: exon numbering in Guicheney et al. [1998] skipped exon 6.

RNA

PTT

Legend:
Eight overlapping  primers covering most (~97%) of the LAMA2c DNA coding sequence (Pegoraro et al. [1998]). After RT-PCR, these primers are used in a nested PCR reaction. Subsequently, PCR products are transcribed and translated and synthesized protein products are analysed on PAA-gel. Exonic sequences are in upper case, intronic and gene flanking sequences in lower case and added primer tails in italics. Amplified: region amplified. Numbering of exons is according to the cDNA Reference Sequence. Length: length of PCR-product in basepairs. Forward primer / reverse primer: sequence of forward and reverse primer. Name: name of the primers. [T7] = ggatcc-TAATACGACTCACTATAGGG-agaccaccatg (BamHI cloning site, T7 promoter sequence and translation initiation sequence resp.).

RT-PCR + SSCA

Legend:
All primer sequences from Helbling-Leclerc et al. (1995); after RT-PCR products were analysed using SSCA (3 electrophoresis conditions). Exonic sequences are in upper case, intronic and gene flanking sequences in lower case and added primer tails in italics. Amplified: region amplified. Numbering of exons is according to the cDNA Reference Sequence. Length: length of PCR-product in basepairs. Reference: publication describing the primer(s). Forward primer / reverse primer: sequence of forward and reverse primer. Name: name of the primers.

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