Frequent problems and their resolutions by using thermal asymmetric interlaced PCR (TAIL-PCR) to clone genes in Arabidopsis T-DNA tagged mutants

Figures & data

Table 1. AD primers used in this study.

Primers	Primer sequences (5′–3′)	Tm (°C)	GC (%)	Degeneracy
AD1	NTCGASTWTSGWGTT	39.9	40.00%–46.70%	64
AD2	NGTCGASWGANAWGAA	43.86	37.50%–50.00%	128
AD3	WGTGNAGWANCANAGA	43.86	31.30%–50.00%	256
AD4	WGGWANCWGAWANGCA	48.99	37.50%–50.00%	256
AD5	WCGWWGAWCANGNCGA	51.55	43.80%–56.30%	256
AD6	WGCNAGTNAGWANAA	44	26.70%–46.70%	256
AD7	AWGCANGNCWGANATA	47.71	31.30%–50.00%	256
AD8	SSTGGSTANATWATWCT	48.56	35.30%–41.20%	128
AD9	CGSATSTCSAANAAWAT	48.56	35.30%–41.20%	64
AD10	AGWGNAGWANCAWAGG	46.43	37.50%–50.00%	128
AD11	TGWGNAGWANCASAGA	48.99	37.50%–50.00%	128
AD12	STTGNTASTNCTNTGC	50.27	37.50%–56.30%	256
AD13	CAWCGNCNGANASGAA	52.83	43.80%–62.50%	256
AD14	TCSTNCGNACNTWGGA	52.83	43.80%–62.50%	256
AD15	WCAGNTGWTNGTNCTG	50.27	37.50%–56.30%	256
AD16	TCTTNCGNACNTNGGA	51.55	37.50%–62.50%	256
AD17	TTGNAGNACNANAGG	48.1	33.30%–60.00%	256
AD18	GTNCGASWCANAWGTT	48.99	37.50%–50.00%	128
AD19	NTCAGSTWTSGWGWT	46.73	40.00%–46.70%	128
AD20	TCNGSATWTGSWTGT	46.73	40.00%–46.70%	64
AD21	NCASGAWAGNCSWCAA	51.55	43.80%–56.30%	256
AD22	NTSGASNTCNGAATCA	50.27	37.50%–56.30%	256

Table 2. Nested specific primers used in this study.

Primers	Primer sequences (5′–3′)	Tm (°C)	GC (%)	Vector
LBa1	TGGTTCACGTAGTGGGCCATCG	63.8	59.10%	PROK2
LBb1.3	ATTTTGCCGATTTCGGAAC	53.25	42.10%	PROK2
LBb1	GCGTGGACCGCTTGCTGCAACT	65.66	63.60%	PROK2
RB1	GTCTGTTGTGCCCAGTCATAG	59.97	52.40%	PROK2
RB1.5	GACCTTAGGCGACTTTTGAACG	60.07	50.00%	PROK2
RB2	ACGGCTTGTCCCGCGTCATC	63.95	65.00%	PROK2
RB3	TGTCGTTTCCCGCCTTCAGT	59.85	55.00%	PROK2
RB4	ATTGGCGGGTAAACCTAAGAGA	58.21	45.50%	PROK2
RB5	GAGAAAAGAGCGTTTATTAGAA	52.62	31.80%	PROK2
LB0	TTCTCATCTAAGCCCCCATTTGGAC	61.98	48.00%	pSKI015
LB1	ATACGACGGATCGTAATTTGTC	56.35	40.90%	pSKI015
LB2	TAATAACGCTGCGGACATCTAC	58.21	45.50%	pSKI015
LB3	ACCATCATACTCATTGCTGATCC	58.4	43.50%	pSKI015
LB3'	TTGACCATCATACTCATTGCTG	56.35	40.90%	pSKI015
ds5-0.5	GAGAGAGGCAGAGCAGCGTTC	63.9	61.90%	PWS31
ds5-0.6	GGTTATGGATGGGAGTTGGAG	60	52.40%	PWS31
ds5-1	ACGGTCGGGAAACTAGCTCTAC	61.9	54.50%	PWS31
ds5-2	CCGTTTTTGTATATCCCGTTTCCGA	60.3	44.00%	PWS31
ds5-3	TACCTCGGGTTCGAAATCGAT	58	47.60%	PWS31
ds5-4	TAGCATAACGGTACGGTACGG	60	52.40%	PWS31
ds3-1	ACCCGACCGGATCGTATCGGT	63.87	61.90%	PWS31
ds3-2	CGATTACCGTATTTATCCCGTTC	58.4	43.50%	PWS31
ds3-3	GTATTTATCCCGTTCGTTTTCGT	56.6	39.10%	PWS31
ds3-4	CCGTCCCGCAAGTTAAATATG	58	47.60%	PWS31

Table 3. Settings for TAIL-PCR.

			Reaction system^c
Steps	Cycles	Thermal programs	Components	Volume (μL)
TAIL-1	1	95 °C, 2 min	ddH2O	32.5
	5	94 °C, 30 s; 67 °C, 1 min; 72 °C, 2.5 min	10 ×  PCR buffer	5
	1	94 °C, 30 s; 25 °C, 3 min; ramping to 72 °C, 0.3 °C/s; 72 °C, 2.5 min	dNTP (2.5 mmol/L)	4
			Nested specific primer 1 (10 μmol/L)^b	1
	15	94 °C, 10 s; 67 °C, 1 min; 72 °C, 2.5 min	rTaq™ (5 U/μL)	0.5
		94 °C, 10 s; 67 °C, 1 min; 72 °C, 2.5 min	Template (10–20 ng/μL)	2
		94 °C, 10 s; 44 °C, 1 min; 72 °C, 2.5 min	ADX primer (10 μmol/L)^a	5
	1	72 °C, 5 min	Total	50
TAIL-2	1	95 °C, 2 min	The same as the primary PCR reaction except for:	47
	15	94 °C, 10 s; 64 °C, 1 min; 72 °C, 2.5 min	Nested specific primer 2 (10 μmol/L)^b	1
		94 °C, 10 s; 64 °C, 1 min; 72 °C, 2.5 min	Template (100-fold diluted TAIL-1 PCR product)	2
		94 °C, 10 s; 44 °C, 1 min; 72 °C, 2.5 min	Total	50
	1	72 °C, 5 min
TAIL-3^d	1	95 °C, 2 min	The same as the primary PCR reaction except for:	47
	20	94 °C, 15 s; 44 °C, 1 min; 72 °C, 2.5 min	Nested specific primer 3 (10 μmol/L)^b	1
	1	72 °C, 5 min	Template (10-fold diluted TAIL-2 PCR product)	2
			Total	50

a ADX represent one of the 22 AD primers in Table 1.

b Nested specific primers in Table 2.

c To reduce cost, the reaction system can be appropriately reduced.

d TAIL-3 PCR products were sequenced.

Figure 1. Four T-DNA insertion sites were cloned by TAIL-PCR in the v1 mutant. AD1-1 and AD1-2 denote two individual DNA fragments amplified by using AD1. Brackets indicate the length of the flanking sequence.

Figure 2. Flanking sequences cloned by TAIL-PCR. Arrows represent the position of the nested specific primers in the T-DNA border. Brackets show the length of flanking sequence. ×10-Lb2 represents the flanking sequences cloned by using three nested primers Lb0, Lb1 and Lb2 in the ×10 mutant. ×10-Lb3 represents the sequences cloned by using Lb1, Lb2 and Lb3 in the ×10 mutant. di2-Lb2 represents the sequences cloned by using Lb0, Lb1 and Lb2 in the di2 mutant.

Figure 3. Head-to-head insertion mode in the v1 mutant (A). Abnormal gene structure in the dt1 mutant (B).

Figure 4. Partial deletion of two genes in the w52 mutant (A). Deletion of 39 genes in the kd361 mutant (B).