DNA barcoding

DNA Barcoding

Introduction

Project partners

Project rationale

Phase 2 Protocols

Phase 2 Update

Barcoding websites

Phase 2 protocols

Regions and Primers

Five plastid regions were chosen to continue into phase 2, with a sixth region, ndhJ, being added during the pilot stage of phase 2. Each region had an initial set of two forward and two reverse primers:

Gene	Primer	Direction	Sequence 5'-3'
matK	2.1	f	CCTATCCATCTGGAAATCTTAG
	2.1a	f	ATCCATCTGGAAATCTTAGTTC
	5	r	GTTCTAGCACAAGAAAGTCG
	3.2	r	CTTCCTCTGTAAAGAATTC
rpoC 1	1	f	GTGGATACACTTCTTGATAATGG
	2	f	GGCAAAGAGGGAAGATTTCG
	3	r	TGAGAAAACATAAGTAAACGGGC
	4	r	CCATAAGCATATCTTGAGTTGG
rpoB	1	f	AAGTGCATTGTTGGAACTGG
	2	f	ATGCAACGTCAAGCAGTTCC
	3	r	CCGTATGTGAAAAGAAGTATA
	4	r	GATCCCAGCATCACAATTCC
accD	1	f	AGTATGGGATCCGTAGTAGG
	2	f	GGRGCACGTATGCAAGAAGG
	3	r	TTTAAAGGATTACGTGGTAC
	4	r	TCTTTTACCCGCAAATGCAAT
YCF5	1	f	GGATTATTAGTCACTCGTTGG
	2	f	ACTTTAGAGCATATATTAACTC
	3	r	ACTTACGTGCATCATTAACCA
	4	r	CCCAATACCATCATACTTAC
ndhJ	1	f	CATAGATCTTTGGGCTTYGA
	2	f	TTGGGCTTCGATTACCAAGG
	3	r	ATAATCCTTACGTAAGGGCC
	4	r	TCAATGAGCATCTTGTATTTC

Following the initial phase 2 pilot screening, where the six regions were tested in representative taxa from each of the study groups for ease of amplification and level of variability, the decision was taken to exclude YCF5 from the remainder of phase 2. Whilst YCF5 was potentially useful for some plants, it failed to yield sufficiently high quality sequences for certain taxonomic groups and has apparently been deleted from the plastid genome of bryophytes. It was therefore felt that YCF5 did not reach the requirements for a suitable barcode for all land plants.

Amplification Conditions

PCR reaction:

PCR mix

Buffer X 1

Mg2+ 1.5mM

dNTPs 0.2mM

FW test primer 1micromolar

RE test primer 1micromolar

Taq DNA polymerase 2 units

BSA 0.1mg/ml

Template (variable)

Water to 20 microlitre

Thermal cycler program:

PCR
94ºC	1min	1 cycle
94ºC	30sec	Variable up to 40 cycles
53ºC	40sec
72ºC	40sec
72ºC	5min	1 cycle

Cycle sequencing is being performed according to the protocols of each partner institute on an extensive sampling of the groups listed.

During the course of phase 2, design of additional primer pairs and modification of the standard protocols outlined above was necessary for some regions. These details are now available as part of the phase 2 update.

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