TABLE 1.

PCR primers used in this study for analysis of CUSφ and control ORFs

ORFa	PCR primers (5′ → 3′)b		Product size (bp)c
	Forward	Reverse
ORF1	CGGGAAGTCTGATTTTGATG	AATGGTGGCGTTCTATCG	308
ORF3	GGGCAACACATCTCTACATTGACG	TTTTGACCAGTCCAGTAAACCAGC	433
ORF5	GAGATAGCCTAAGTTCCGACATCG	CTTCATTTCCCGCAGGTATTCC	452
ORF6	CGGAGAGTTGTGACGAACATTTATGG	CTGGCGTTTATCTGTCTGCTTTTC	539
ORF7	ATGTGCTTTCGCAGGGTCAGAG	CACTTCATCACTCATCGTATCCAGC	516
irp-2	GTTGCTGTCCATCAAGCACG	GCCGGAAAGCCTGGCCTTTA	1,243
AltORF1	CATGTAGAGAACGAATGTGATAACC	CCGCAAAGAATCTCAGATAACATT	397
pyk	GAAGTCACTGAACACACCGTTGTC	ACCGTCAAGGATGGCGTTTG	509

^aORF3, ORF5, ORF6, and ORF7 are present in both CUS-1 and CUS-2 as defined in the text, whereas AltORF1 of Y. pestis CUS-2 is located in the same relative genetic location as ORF1 but is not homologous. Note that the puvA (ORF5) primers do not flank the insertion in the previously described puvA::kan mutant (6).

^bPrimers were purchased from IDT (Coralville, Iowa) and were used in PCRs at a final concentration of 8.75 pM 20-μl (total volume) mixtures containing PCR master mixture and MgCl₂ purchased from Promega (Madison, Wis.). Amplification was carried out with a PTC-200 DNA Engine (MJ Research, Indian Valley, Nev.). The reaction conditions (35 cycles) for PCR amplification of ORF3, ORF5, ORF6, and ORF7 included denaturation at 94°C for 30 s, annealing at 61°C for 30 s, and extension at 72°C for 30 s. The annealing temperature was increased to 51°C for ORF1, pyk of Y. pestis, and irp-2.

^cAmplicons (products) were analyzed by electrophoresis in 0.7% agarose buffered with 40 mM Tris-acetate and 1 mM EDTA. The marker used was the 1-kb ladder purchased from Promega.