Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides

Koh, Chong Mei John; Liu, Yanbin; Moehninsi; Du, Minge; Ji, Lianghui

doi:10.1186/1471-2180-14-50

Table 4 Oligonucleotides used

From: Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides

Name	Sequence (5′ to 3′)	Specificity
Rg70f3	GTTCATCCCGAGGACCATCAGTC	Sense primer for KU70 3′ RACE Specific primer for ∆ku70 fungi colony PCR
Rg70r3	GGGATGAGCTCCTTGAGCTTTGC	Antisense primer 1 for KU70 5′ RACE
Rg70r4	TCTTCGTCCGGGTAGATGAAGTAG	Antisense primer 2 for KU70 5′ RACE
Rg70r5	ATCCTTTGGGCTGGCGATGACTTTG	Antisense primer 3 for KU70 5′ RACE
Rg80f2	GTCGCTTGAGCGCATCTTCAACAAG	Sense primer for KU80 3′ RACE
Rg80r2	ATCACGATGTCCTCGTCCTCGTCGT	Antisense primer 1 for KU80 5′ RACE
Rg80r3	CTCCAACCTGCGGACATGCTTCTT	Antisense primer 2 for KU80 5′ RACE
Rg80r4	GAAGCGAGCCTTGCGCTTGATCTCG	Antisense primer 3 for KU80 5′ RACE
Rg70Lf	CTCGTGTCAAGAAAACCAGCAAG	KU70 gene deletion region
Rg70Rr	ACCTCAGCAACCAACCTACCAGT	KU70 gene deletion region
C50f	TCCTCCTACCGACGCTCTACC	CAR2 deletion region (50 bp homology length)
C50r	CTCTGTGCAGCCATCTCGTTC	CAR2 deletion region (50 bp homology length)
C100f	TTCATTGCCTCAACCCACATC	CAR2 deletion region (100 bp homology length)
C100r	CGTCCCCAATGCTGAGATAAA	CAR2 deletion region (100 bp homology length)
C250f	CAAAGTTGGAGGAGGGAGGAG	CAR2 deletion region (250 bp homology length)
C250r	GTTGAGAAACCAGCGAGACCA	CAR2 deletion region (250 bp homology length)
C500f	CCTGGGACTCGTACCTCATCC	CAR2 deletion region (500 bp homology length)
C500r	GCGACCGCGACTGACTAGTTT	CAR2 deletion region (500 bp homology length)
Rt079	GGACTGGACTACTGGCTCGTGT	CAR2 deletion region (750 bp homology length)
Rt080	TCAAGAGCTACCAGGAGAGCAAC	CAR2 deletion region (750 bp homology length) CAR2 complementation region
C1000f	GGGGCTGTCTGTGACTTGCTAT	CAR2 deletion region (1000 bp homology length)
C1000r	TCGAGCTCAAGCTGAAGAAGAAG	CAR2 deletion region (1000 bp homology length)
C1500f	CTCTCGCACGGGGAGGTAGT	CAR2 deletion region (1500 bp homology length) CAR2 complementation region
C1500r	TGCTTGCCAAGAAGCCTACAATA	CAR2 deletion region (1500 bp homology length)
STE20Lf	GCTCGTGTTGCGGGAACAGT	STE20 deletion region
STE20Rr	GGGCACGCCTTCTTCAAGAAC	STE20 deletion region
Rt033	GTCAAGCCGCCCAGGCTGTC	URA3 deletion region
Rt034	GGATCCGCCAAGTCGCGCAG	URA3 deletion region
Rg70r2	GTCCTTCTTCACCACGCGTTTCTT	Specific primer for ∆ku70 fungi colony PCR
Rt096	CGAATCCAACATCGACATGATTT	Specific primer for ∆car2 fungi colony PCR
Rt097	TATTGACGAGCTGAAGAGCCTGT	Specific primer for ∆car2 fungi colony PCR
Rt006	TCTCCCTCGCCCTCTGCT	Specific primer for GPD1 (reference gene) in multiplex PCR
Rt007	AGCCATGCCGGTGAGCTTG
Rt100	GACCCGACAGACGAGTGGAC	KU70 probe for Southern blot
Rt101	AGGTCCTTGAGAAAGCGGATG	KU70 probe for Southern blot
Rt083	CTCACCCTCGTGTTGCTCGTA	CAR2 probe for Southern blot
Rt084	CCTCTCCTCCCTCCTCCAACT	CAR2 probe for Southern blot

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ISSN: 1471-2180

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