BMC Genomics

Table 5 Pre-restriction of immobilized cDNA

From: Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA

	No pre-restriction		Partial pre-restriction		Full pre-restriction
*Arabidopsis thaliana*
Marking enzymes	FatI	MboI	HinPI	MboI	FatI	MboI
Releasing enzymes	MboI	FatI	MseI	MseI	MboI	FatI
	MseI	MseI	TasI	TasI	MseI	MseI
	TasI	TasI	TaqI	TaqI	TasI	TasI
Sequence coverage	78 %		70 %		52 %
Sequence redundancy	30 %		30 %		0 %
Average fragment length (nt)	183		237		142
PCR reactions	100 %		125 %		36 %
Mouse
Marking enzymes	MboI	Csp6I	HinPI	MboI	MboI	Csp6I
Releasing enzymes	Csp6I	MseI	TasI	TasI	Csp6I	MseI
	MseI	MboI	MseI	MseI	MseI	MboI
	FatI	FatI	TaqI	TaqI	FatI	FatI
Sequence coverage	87 %		81 %		50 %
Sequence redundancy	41 %		35 %		0 %
Average fragment length (nt)	270		271		159
PCR reactions	100 %		39 %		25 %
Human
Marking enzymes	MboI	Csp6I	HinPI	MboI	MboI	Csp6I
Releasing enzymes	CspI	MboI	MseI	MseI	CspI	MboI
	HinPI	FatI	TasI	TasI	HinPI	FatI
	FatI	HinPI	TaqI	TaqI	FatI	HinPI
Sequence coverage	85 %		84 %		63 %
Sequence redundancy	35 %		40 %		0 %
Average fragment length (nt)	274		292		182
PCR reactions	100 %		86 %		61 %

Simulation of cDNA-AFLP with sequential digestion protocol on Arabidopsis, human and mouse RefSeq ESTs was performed. Optimal combination of restriction enzymes (2 marking in combination with 3 releasing enzymes) with the highest sequence coverage was used to compare the effect of partial pre-restriction and full pre-restriction options on maximal transcript coverage, sequence redundancy, average fragment length and reduction of PCR effort (relative to the maximal number of PCR by one of the options).

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ISSN: 1471-2164

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