Mutagenic polymerase chain reaction strategy and allele-specific restriction enzyme digestions used to perform messenger RNA analysis
| Amino acid change (nucleotide change) . | Primer couple* 5′.3′ . | Position† . | Exon . | PCR product (bp)‡ . | Restriction enzyme . | Fragment size wt/mut (nt)1-153 . |
|---|---|---|---|---|---|---|
| Gly13stop | F:AGATGTTTTCCATGAGGATCGTCTGCCTAGT | 29 -59 | 1 | 126 | FokI | 106+20/126 |
| (1193G>T) | R:TTTCCACAACCCTTGGGCCACGCACGC ATC | 1223 -1194 | 2 | |||
| FS51stop | F:AGATGTTTTCCATGAGGATCGTCTGCCTAGT | 29 -59 | 1 | 388/387 | BstXI | 388/269+118 |
| (1215delT) | R:CGGTTGTAGGTATTATCACGGTTATTGGCT | 3076 -1915 | 3, 4 | |||
| Ser100stop | F:GCAGGATGAAAGGGTTGATTGATGAAGTCA | 1761 -1790 | 3 | 184 | FokI | 135+44/179 |
| (1914C>G) | R:CGGTTGTAGGTTTATCACGGTTATTGG AT | 3076 -1915 | 3, 4 | |||
| Arg110stop | F:AGATGTTTTCCATGAGGATCGTCTGCCTAGT | 29 -59 | 1 | 417 | HpaII | 386+31/417 |
| (3075C>T) | R:CAATTCTGCTTCTCAGATCCTCTGACAC CC | 3105 -3076 | 4 | |||
| Arg149stop | F:TGTTAGAGCTCAGTTGGTTGATATG TAA | 3164 -3191 | 4 | 147 | MaeIII | 118+29/147 |
| (3192C>T) | R:ACCTGTTCAAGTTGCTTCTGCTG | 3895 -3873 | 5 |
| Amino acid change (nucleotide change) . | Primer couple* 5′.3′ . | Position† . | Exon . | PCR product (bp)‡ . | Restriction enzyme . | Fragment size wt/mut (nt)1-153 . |
|---|---|---|---|---|---|---|
| Gly13stop | F:AGATGTTTTCCATGAGGATCGTCTGCCTAGT | 29 -59 | 1 | 126 | FokI | 106+20/126 |
| (1193G>T) | R:TTTCCACAACCCTTGGGCCACGCACGC ATC | 1223 -1194 | 2 | |||
| FS51stop | F:AGATGTTTTCCATGAGGATCGTCTGCCTAGT | 29 -59 | 1 | 388/387 | BstXI | 388/269+118 |
| (1215delT) | R:CGGTTGTAGGTATTATCACGGTTATTGGCT | 3076 -1915 | 3, 4 | |||
| Ser100stop | F:GCAGGATGAAAGGGTTGATTGATGAAGTCA | 1761 -1790 | 3 | 184 | FokI | 135+44/179 |
| (1914C>G) | R:CGGTTGTAGGTTTATCACGGTTATTGG AT | 3076 -1915 | 3, 4 | |||
| Arg110stop | F:AGATGTTTTCCATGAGGATCGTCTGCCTAGT | 29 -59 | 1 | 417 | HpaII | 386+31/417 |
| (3075C>T) | R:CAATTCTGCTTCTCAGATCCTCTGACAC CC | 3105 -3076 | 4 | |||
| Arg149stop | F:TGTTAGAGCTCAGTTGGTTGATATG TAA | 3164 -3191 | 4 | 147 | MaeIII | 118+29/147 |
| (3192C>T) | R:ACCTGTTCAAGTTGCTTCTGCTG | 3895 -3873 | 5 |
PCR indicates polymerase chain reaction; bp, base pairs; wt, wild type; nt, nucleotide; mut, mutation; and FS, frameshift.
Mutated nucleotides are double-underlined.
Product lengths refer to PCR amplifications performed on complementary DNA.
Lengths of the restriction fragments detectable by the automated DNA sequencer are underlined.