Extension primers
All CATMA GSTs are flanked by one of 24 5' extension primers and one of 16 3' extensions. These allow reamplification of the full set of CATMA GSTs using just 40 primers, while reducing the risk of cross-contamination by ensuring that every GST in a 384-well microtitre plate has a different pair of extension primers. The primer design parameters and sequences are described below.
Design parameters
- All 17mers
- GC content of ~50% (ie 8 or 9 of the 17 bases)
- No hairpins (self-complementary repeats of 4 or more bases separated by three or more bases)
- No self primer-dimers (last 4 bases must not base-pair anywhere within the primer)
- No runs of more than 4 G/Cs (eg GGCC is OK, but GGCGC is rejected)
- BLAST search against the genome sequence BACs must give no matches under the conditions used - this means no more than 14 of the 17 bases matching in a non-gapped BLAST (and no more than 13 in a row). More stringent than this is not really possible with a genome of the size of Arabidopsis.
- No primer-dimers between any of the row primers and any of the column primers (i.e. last 4 bp matching anywhere in the other primers).
- No stop codons or start codons within the primers (sense orientation)
All primers have also been tested to minimise priming against the BAC plasmid backbone and E. coli genomic DNA.
Primer sequences
C1 CTTACGTTCTGTCGGAC C13 TTCTACCGGACCTACCA C2 AGGTATACACGCACAGC C14 AAGATACGGCACGCTAC C3 GGCTTTACAGTTACGGG C15 GTCCGCTGTATACGAGA C4 TTGCCAAGAGGGTCTAC C16 GCGAGTTCGTACGACAT C5 CACCTCGGATCGTTTTA C17 TACCCTGTGTCCCGTTT C6 CTCACGCGATACGAATC C18 CGCGTATTGCGAGAGTA C7 AGCGACGGCATTCTATC C19 CCTACGGTATCGTGTCA C8 GTTATACGACGGACAGC C20 CCGATATCCAGTCAGGA C9 CCTGTGCGACGTTATCT C21 CGCGTTCTATCCTACAG C10 AAGGGAGTCCGGTGTAT C22 GTACGCGTTACACGAGT C11 GCCAAGTTGCGTGTCTA C23 CCCTACGGCAATTTCGT C12 TATACGAATTCCTGGGG C24 GGGTCCTATCTGGTCAT R1 TATCCGAGCGTCAGCTA R9 GGCGAAGCGTTCCATTA R2 CCACTTGGGCACACTAT R10 TACCTTACCGACCGAGT R3 GGGTACCTTCGCATTAC R11 CGCTACAGCGCACTTTA R4 TGCGCAATCGTTCAGTC R12 GCGCACAGGTTGTCTAT R5 GTACCAGCGGTTCATTA R13 ACGCCTTCATTCGTCTA R6 TACCGTATTGCCGAAGG R14 TGCGACGTCAATTCAGT R7 TGGCAACGTGCCCTTAT R15 CGCAGCCAGGTGTATTA R8 TACCCAGACTTCCGTTC R16 GTGCCTTCGACAGACTA R = row primers, C = column primers.
These sequences can be downloaded as a Microsoft Word file.
These sets have been chosen to allow reamplification of GSTs from a 384-well microtitre plate using a single primer for each row and each column, while ensuring that each well on a plate requires a unique combination of extension primers. Please see the diagram below:
Initial GST synthesis was carried out on 96-well plates, each of which required only twelve column and eight row extensions. Four combinations of extensions will be used to produce the desired arrangement on 384-well plates:
1 - C1-C12, R1-R8 2 - C13-C24, R1-R8 3 - C1-C12, R9-R16 4 - C13-C24, R9-R16Following transfer to a 384-well plate, the rows and columns should occur in the following order:
C1 C13 C2 C14 C3 C15 ...
R1
R9
R2
R10
...
In addition, the GSTs are all oriented so that the column primer always
lies on the sense strand of the gene, while the row primer always lies
on the antisense strand. This is irrespective of the orientation of the
gene in the chromosome.