BioChip J. (2012) 6(4): 325-334DOI 10.1007/s13206-012-6404-0

Abstract Countless species occur in the marinemicroalgal domain. Some are used as health func-tional foods or medical products but many species areharmful such as those that cause the red tide. There-fore, it is necessary to conduct prompt and accurateidentification of microalgal species. As it is quitedifficult to accurately distinguish all species in termsof morphology, we performed DNA barcoding analy-sis using molecular markers for more accurate andrapid screening. DNA barcoding analysis, i.e., DNAchip technology, is a powerful method for studies onmicroalgal taxonomy and biodiversity. We used themitochondrial cytochrome c oxidase subunit I (mtCOI)as a barcoding gene to identify microalgal species. Inthis study, the diversity and phylogenetic differencesamong different microalgae were analyzed. Additio-nally, a microalgal species-specific probe was screen-ed by 21-23 bp and the result was printed on silylatedslide for use in a robotic microarrayer. As a result, weperformed a DNA chip assay for each of 25 microalgal

species and determined that the COI barcode genewas suitable as a marker gene, as it could identifyvarious microalgae from the Korean South Sea byspecies.

Keywords: Microalgae, DNA barcoding, Mitochond-rial cytochrome c oxidase subunit I, DNA chip assay,Marker gene

Introduction

Marine microalgae are primary producers and usefulbiological resources, as they provide the food basefor bio-organisms used for culturing, pharmaceuticalproducts, and biological fertilizers. However, rapidproliferation of some marine microalgae may causetremendous harm to the marine ecosystem and fish-ery resources. The rapid proliferation of some marinemicroalgae creates red tides, which leads to death ofmany organisms and causes harmful algal blooms bydischarging toxic substances into the sea. Therefore,early identification of marine microalgal species shouldbe prioritized to take a speedy response against thesered tides1,2.

Ways to identify species using morphological charac-ters include general biological appearance, specificstructure, internal morphology, and embryological dif-ferences. Additionally, characters such as the physio-logy characters of metabolism and excretion, behavio-ral characters such as courtship displays, and ecologi-cal characters such as habitats, food, seasonal variation,and biogeological distribution characters3,4 are usedto identify species.

Many methods are available to classify life using

Original Research

Development of DNA chip for verification of 25 microalgaecollected from southern coastal region in Korea

Gunsup Lee1, So Yun Park2, Seungshic Yum1, Seonock Woo1, Youn-Ho Lee3, Seung Yong Hwang4,Heung-Sik Park5, Sang Hyun Moh6, Sukchan Lee7 & Taek-Kyun Lee1

Received: 21 July 2012 / Accepted: 13 August 2012 / Published online: 20 December 2012�The Korean BioChip Society and Springer 2012

1South Sea Environment Research Department, Korea Institute ofOcean Science and Technology, Geoje 656-830, Korea2Medical Research Center of Neural Dysfunction, GyeonsangNational University, Jinju 660-751, Korea3Marine Living Resources Research Department, Korea Institute ofOcean Science and Technology, Ansan, Gyeonggi-do 425-600, Korea4Department of Biochemistry, Hanyang University & GenoCheck Co.Ltd., Sangnok-gu, Ansan, Gyeonggi-do 426-791, Korea5Korea South Pacific Ocean Research Center, Korea Institute ofOcean Science and Technology, Ansan, Gyeonggi-do 425-600, Korea6Bio-FD&C., Ltd, Anti-aging Research Institute, 57 Eunbong-ro,Namdong-gu, Incheon 405-849, Korea7Department of Genetic Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, KoreaCorrespondence and requests for materials should be addressed to T.-K. Lee ( tklee@kiost.ac)

molecular biological characters. A worldwide consor-tium was established called The Consortium for theBarcode of Life with the purpose of unifying species

identification methods using a genetic barcode5. Thegene used as the genetic barcode was the mitochondrialcytochrome c oxidase subunit I (COI). Several studies

326 BioChip J. (2012) 6(4): 325-334

Figure 1. Sequence alignment of mtCOI genes from 25 microalgal species (Achnanthes longipes, Amphora sp., Asterionella gla-cialis, Chaetoceros atlanticus, Chaetoceros didymus, Chaetoceros septentrionalis, Chaetoceros vistulae, Chlorella ellipsoidea,Chlorophyta UF, Coscinodiscus perforatus, Cylindrotheca closterium, Cylindrotheca fusiformis, Cymatosira lorenziana, Dity-lum brightwellii, Gloeocystis gigas, Gyrodimium impudicum, Heterosigma akashiwo, Melosira nummuloides, Navicula sp., Nitz-schia pungens, Nitzschia subpacifica, Prorocentrum minimum, Skeletonema costatum, Stephanopyxis turris, Thalassiosira allenii).

BioChip J. (2012) 6(4): 325-334 327

Tab

le 1

.Nuc

leot

ide

iden

titie

s(up

per)

and

div

erge

nce

(low

er)

of m

icro

alga

l spe

cies

.

%1

23

45

67

89

1011

1213

1415

1617

1819

20

1-

60.8

61.4

60.8

60.8

60.7

62.8

64.4

62.8

60.9

61.8

61.8

62.2

59.7

59.4

65.2

62.2

61.8

61.1

59.5

257

.1-

74.4

75.8

74.5

74.5

73.7

75.5

54.1

78.3

79.9

80.3

61.6

75.8

78.7

75.9

72.0

72.5

74.2

75.9

355

.031

.4-

83.1

80.7

81.8

81.4

76.2

55.4

79.4

75.6

75.1

60.1

83.1

74.6

75.5

72.7

74.4

80.1

72.0

456

.329

.319

.2-

85.1

82.5

85.1

76.3

55.5

80.3

75.6

76.3

60.3

85.9

77.0

76.9

72.0

77.9

82.0

73.6

556

.631

.222

.316

.7-

80.7

83.4

76.2

54.7

79.4

75.8

75.1

60.3

83.5

74.4

76.4

69.7

79.4

77.3

71.1

656

.631

.220

.919

.922

.3-

82.0

75.5

55.3

78.8

77.9

77.0

61.7

85.6

75.4

75.5

72.3

76.3

80.0

73.0

752

.032

.521

.516

.718

.920

.7-

75.6

55.5

80.4

75.5

76.1

61.0

83.5

74.9

74.8

71.8

76.8

80.3

73.3

849

.129

.728

.728

.428

.629

.829

.5-

57.7

76.9

76.5

75.1

63.2

77.5

75.1

80.8

72.1

74.2

74.4

72.9

951

.572

.468

.467

.870

.068

.267

.662

.4-

53.9

55.3

55.7

68.4

55.3

53.1

58.1

57.1

55.0

55.5

54.2

1056

.025

.624

.122

.924

.124

.922

.827

.772

.3-

79.4

77.2

62.2

83.2

77.0

76.0

70.9

79.0

77.7

75.1

1154

.823

.529

.529

.529

.326

.229

.728

.369

.224

.1-

82.8

64.0

79.5

80.3

75.7

75.1

73.5

75.5

77.7

1254

.822

.930

.428

.530

.427

.428

.930

.367

.827

.319

.5-

61.1

77.4

79.7

75.6

73.2

73.5

74.9

77.0

1353

.454

.157

.056

.756

.753

.655

.350

.841

.652

.649

.155

.0-

62.9

61.8

60.2

62.0

55.8

59.7

61.1

1458

.829

.219

.215

.618

.716

.018

.726

.768

.619

.123

.926

.951

.2-

77.5

76.9

73.2

77.8

82.5

74.6

1559

.625

.031

.027

.431

.429

.930

.630

.474

.327

.522

.923

.653

.326

.7-

77.9

73.9

71.0

75.1

75.7

1647

.729

.229

.827

.728

.329

.730

.922

.261

.629

.029

.429

.556

.927

.526

.3-

72.8

73.3

74.3

72.2

1752

.735

.134

.135

.138

.934

.935

.434

.963

.936

.830

.333

.153

.333

.332

.133

.9-

68.3

69.4

67.7

1854

.134

.231

.426

.324

.228

.627

.931

.668

.924

.832

.832

.866

.926

.436

.733

.041

.2-

74.8

70.4

1955

.731

.623

.120

.727

.023

.323

.031

.467

.626

.529

.730

.657

.920

.030

.331

.539

.330

.8-

72.4

2060

.029

.235

.132

.636

.533

.533

.133

.871

.230

.326

.527

.554

.930

.929

.434

.742

.337

.634

.4-

2150

.836

.123

.924

.326

.425

.825

.632

.866

.326

.231

.936

.156

.424

.033

.633

.536

.031

.521

.440

.722

10.5

57.7

55.0

57.8

56.5

55.6

52.6

48.8

51.5

56.6

55.7

55.2

53.6

58.2

60.1

47.3

52.1

53.0

56.2

59.8

2352

.632

.120

.719

.221

.020

.321

.130

.065

.823

.929

.830

.855

.217

.632

.731

.240

.329

.424

.334

.224

54.7

29.3

27.1

21.8

23.7

25.1

26.8

30.1

72.1

22.1

25.6

28.2

57.9

21.8

30.1

28.5

36.7

21.7

26.7

32.6

2549

.358

.461

.257

.359

.558

.557

.154

.742

.159

.356

.955

.741

.056

.361

.056

.454

.961

.959

.262

.8

1, A

chna

nthe

s lo

ngip

es;

2, A

mph

ora

sp.;

3, A

ster

ione

lla

glac

iali

s; 4

, C

haet

ocer

os a

tlan

ticu

s; 5

, C

haet

ocer

os d

idym

us;

6, C

haet

ocer

os s

epte

ntri

onal

is;

7, C

haet

ocer

os v

istu

lae;

8,

Chl

orel

lael

lips

oide

a; 9

, Chl

orop

hyta

UF

; 10

, Cos

cino

disc

us p

erfo

rate

s; 1

1, C

ylin

drot

heca

clo

ster

ium

; 12

, Cyl

indr

othe

ca f

usif

orm

is;

13, C

ymat

osir

a lo

renz

iana

; 14

, Dit

ylum

bri

ghtw

elli

i; 15

, Glo

eocy

stis

giga

s; 1

6, G

yrod

imiu

m i

mpu

dicu

m;

17, H

eter

osig

ma

akas

hiw

o; 1

8, M

elos

ira

num

mul

oide

s; 1

9, N

avic

ula

sp.;

20, N

itzs

chia

pun

gens

; 21

, Nit

zsch

ia s

ubpa

cifi

ca;

22, P

roro

cent

rum

min

imum

; 23

,Sk

elet

onem

a co

stat

um; 2

4, S

teph

anop

yxis

turr

is; 2

5, T

hala

ssio

sira

all

enii

2122

2324

25

63.4

98.5

62.7

61.7

64.0

71.4

60.5

73.9

75.8

59.6

79.7

61.4

82.0

77.3

58.3

79.3

60.3

83.1

81.1

60.0

77.9

60.8

81.7

79.7

59.0

78.3

61.1

82.3

78.7

59.4

78.5

62.5

81.7

77.6

60.1

73.5

64.5

75.4

75.2

61.2

56.1

62.8

56.4

53.8

67.9

78.0

60.6

79.5

81.0

59.1

74.1

61.4

75.5

78.3

60.1

71.4

61.7

74.8

76.5

60.7

60.4

62.0

61.0

59.7

68.5

79.5

60.0

84.3

81.1

60.5

73.0

59.3

73.5

75.2

58.3

73.2

65.3

74.5

76.3

60.5

71.4

62.5

68.9

71.0

61.2

74.4

62.2

75.8

81.3

57.8

81.4

61.0

79.3

77.6

59.1

68.7

59.5

72.6

73.6

57.5

-63

.479

.976

.559

.450

.9-

62.0

61.1

63.2

23.6

54.2-

78.5

59.6

28.3

56.0

25.4-

58.8

58.5

51.0

58.1

59.9-

have applied the COI gene to identify to plants aswell as fishes in Salmonidae, Rajidae, and Batoideaand marine organisms such as Cephalopoda and Ost-reidae6-9.

Barcoding studies to identify microalgal specieshave been conducted using several genes. In 2006,Lavinia Robba et al. classified 32 kinds of red algae(Rhodophyta) using a polymerase chain reaction (PCR)analysis of COI genes10. In 2005, Alverson and Kol-nick also identified diatom species through PCR ana-lysis using small subunit ribosomal DNA11. In addit-ion to COI, SSU rDNA, and 5.8S++internal transcrib-ed spacer (ITS) 2 were used to distinguish diatom spe-cies12.

We conducted species identifications using the COIgene and also attempted species identification using aDNA chip, which has not been tried in marine micro-algae. As a result, 25 species of microalgae from theKorean coast were verified using DNA chip techno-logy. The species identifications by DNA chip areexpected to be used in the laboratory and also for realtime on-site species identification.

Results and Discussion

Sequence analysis

The mitochondrial COI gene has been used as a gene-tic marker in various organisms. We carried out sequ-ence analyses to identify whether identification waspossible using species markers. Sequence analyseswere conducted using 25 microalgal species (Achnan-thes longipes, Amphora sp., Asterionella glacialis,Chaetoceros atlanticus, Chaetoceros didymus, Chaeto-ceros septentrionalis, Chaetoceros vistulae, Chlorellaellipsoidea, Chlorophyta UF, Coscinodiscus perfora-tes, Cylindrotheca closterium, Cylindrotheca fusifor-mis, Cymatosira lorenziana, Ditylum brightwellii, Glo-eocystis gigas, Gyrodimium impudicum, Heterosigmaakashiwo, Melosira nummuloides, Navicula sp., Nitz-schia pungens, Nitzschia subpacifica, Prorocentrumminimum, Skeletonema costatum, Stephanopyxis turris,Thalassiosira allenii) (Figure 1). The results from theanalysis of the nucleotide composition of 749 bp show-ed that the sequence was composed of A (25%), T(38.7%), G (19.2%), and C (17.1%). Additionally, theproportions of conserved sites were identified; variablesites and singleton sites were 269/749 (35.9%), 480/749 (64.1%), and 60/749 (8%) respectively. The resultsof inter-species identity and diversity revealed thatmost inter-species differences were ⁄85.9% in iden-tity. But, 98.5% were differentiated from the identityresult in Achnanthes longipes and Prorocentrum mini-mum and also there was a small difference of 10.5%

in diversity (Table 1). Similarly, the same inter-spe-cies differences to that of the identity comparison werefound in the phylogenetic analysis (Figure 2). There-fore, using the mitochondrial COI gene was sufficientto distinguish microalgal species.

Probe selection

The mitochondrial COI gene was used to screen for aspecies-specific probe to identify microalgal species.Based on each of the inter-species variation sites, theprobe was produced at a length of 21-23 bp with a dif-ference over 2 bases (Table 2). Achnanthes longipes,Chaetoceros septentrionalis, Cylindrotheca fusiformis,Ditylum brightwellii, Gloeocystis gigas, Gyrodimiumimpudicum, Heterosigma akashiwo, Nitzschia sub-pacifica, Prorocentrum minimum, and Stephanopyxisturris were spotted from two positions by one probe,whereas Amphora sp., Asterionella glacialis, Chaeto-ceros atlanticus, Chaetoceros didymus, Chaetocerosvistulae, Chlorella ellipsoidea, Chlorophyta UF, Cos-cinodiscus perforates, Cylindrotheca closterium, Cy-matosira lorenziana, Navicula sp., Nitzschia pungens,Skeletonema costatum, Thalassiosira allenii were spott-ed from four positions by two probes, and Melosiranummuloides was spotted from six positions by threeprobes (Figure 3b). The sequences of four bases wereadded to the bilateral ends (5′ and 3′) to increase label-ing efficiency (Figure 3a). More detailed informationis presented in Table 2, and the species-specific probeproduced was spotted on a DNA chip.

DNA microarray

The product was labeled with the probe and mountedon the chip, and the Cy3 of each species was perform-

328 BioChip J. (2012) 6(4): 325-334

Figure 2. Topologies obtained via phylogenetic analysis ofmicroalgal COI gene.

ed by hybridization. All species samples showed fluo-rescent signals for each respective specific probe (Fig-ure 4). The signal intensity at the target position wasidentified with high fluorescence values of ≥12,000-

≤65,000 from all 25 microalgal species. In contrast,signal intensity had low values of ≤1,500 for the non-targeted positions. However, Prorocentrum minimumwas identified with higher signal intensity of 7,165.5

BioChip J. (2012) 6(4): 325-334 329

Table 2. Probe selection for microarray analysis.

Species Position RefSeq Start End

Achnanthes longipes 3,4 CACTGCAAAAATCAGATAGAGCG 26 49

Amphora sp. 5,6 GCTCCAGGCTTTTTTATGCATAA 544 5677,8 GAAACTACAGTTCCAATAACACC 55 78

Asterionella glacialis 9,10 GATCCTGTCTTATACCAGCATTT 706 72911,12 CTGGTGCTTCATCAATATTAGGT 485 508

Chaetoceros atlanticus 13,14 AAAGATAGAGCAGTCCCAGCTAC 58 8115,16 CTACTCCAGATATTGCACCAAAA 39 62

Chaetoceros didymus 17,18 TTTTTGACCCTGCAGGGGGTGGA 638 70619,20 CCAGTGCTTGCGGGAGCTATTAC 625 648

Chaetoceros septentrionalis 21,22 GGCAATTACCATGCTTTTAACTG 639 662

Chaetoceros vistulae 23,24 GGCTGTCTTAATAACAGCTTTCT 585 60825,26 TGGCGCAGTGGATTTAGCTATTT 447 470

Chlorella ellipsoidea 27,28 GATCCCGTATTGTACCAACATTT 706 72929,30 GTATGAGTATGCATAGACTACCT 551 574

Chlorophyta UF 31,32 CCCAGTCTTGTTCCAGCACAT 709 73033,34 TGCTGATGGACATCCACTTCG 654 675

Coscinodiscus perforatus 35,36 CAGGTGCTATCACAATGCTTTTA 635 65837,38 TATCGGGAGCTGCTTCTATTTTA 482 505

Cylindrotheca closterium 39,40 CCAGAAATGGCTTGGCATAAATT 547 57041,42 AAATATGCGAAGTCCAGAAATGG 534 557

Cylindrotheca fusiformis 43,44 GGAGGTGATCCAATACTTTATCA 700 723

Cymatosira lorenziana 45,46 CTTTAGTGCAACGGGTGGTGGTG 684 70447,48 GCTTTTGACAGATCGTTTTTACG 651 674

Ditylum brightwellii 49,50 CATCTTTCTGGTGCTTCTTCTAT 478 501

Gloeocystis gigas 51,52 GAGAGCAATAGGAATGACATTTC 540 563

Gyrodimium impudicum 53,54 CTACCTTTTACGATCCGGCAGGA 677 700

Heterosigma akashiwo 55,56 TCCTTGGGCTATCCTTATCAC 580 601

57,58 GATCCCGTTCTTTATCAACATCT 707 729Melosira nummuloides 59,60 TTTTTTTGACCCCGCAGGAGGCG 681 704

61,62 CTGTTCTTGCTGGAGCTATTACT 626 649

Navicula sp. 63,64 GATCCAGTTTTATACCAGCACTT 706 72965,66 GTGTGGTCAGTATTTTTAACAGC 580 603

Nitzschia pungens 67,68 GGAGGAGACCCTATATTATATCA 100 72369,70 GCAGCAGGTATAACTATGTTGTT 634 657

Nitzschia subpacifica 71,72 CTGTGTTATTCCAGCACTTATTC 710 733

Prorocentrum minimum 73,74 CAAAAATGAGATAAAGCGTGCCA 21 44

Skeletonema costatum 75,76 GTCTTGTTTCAGCATCTTTTCTG 712 73477,78 CTGTTTTAGCTGGAGCTATTACA 626 649

Stephanopyxis turris 79,80 CCTTTATTTGCCTGGTCAGTTTT 571 594

Thalassiosira allenii 81,82 CAGCGTTCTTTAATGCTGCTG 678 69983,84 GTTGATTACTGATCGTCACTTTG 651 674

from positions 3 and 4, which were non-target posi-tions (Achnanthes longipes probe) compared to thatof the other species. This occurred because the identitydifference between Prorocentrum minimum and Ach-nanthes longipes was relatively larger, as shown fromthe results of a prior sequence analysis. The same pat-tern as shown in Figure 4 also appeared in the fluores-cence pattern (Figure 5). Chaetoceros septentrionaliscould be identified by detection at both the target andnon-target positions from the results of the fluores-cence pattern (Figure 5). Target positions 21 and 22were identified from the signal intensity results withan arbitrary unit value of 44904.0, whereas the non-target positions 11, 12, 27, and 28 were identified withlower arbitrary unit values of 1184.0 and 1359.0. Addi-tionally, in cases in which it was difficult to identifythe species by fluorescence pattern, the arbitrary unitvalues of Chaetoceros vistulae, Ditylum brightwellii,Skeletonema costatum, and Thalassiosira allenii were14708.0 and 12306.0, 19631.0, 65333.0, and 27940.0as well as 40732.5 and 35102.5, as found from thetarget position respectively, whereas the arbitrary unitvalues of Chaetoceros vistulae, Ditylum brightwellii,Skeletonema costatum, and Thalassiosira allenii wereas low as 1860.0, 2214.0, 3676.0, and 3027.0 as wellas 3182.5, respectively from the non-target position.The results of the fluorescence pattern analysis (Figure

5) revealed that the fluorescence was identifiable fromthe non-target position; therefore, it was determinedthat the species could be identified by a comparisonwith signal intensity (Figure 4).

DNA chip technology is a powerful tool to obtain asignificant amount of accurate information quicklyand conveniently using a small specimen quantity. DNAchip technology, i.e., species differentiation using DNAbarcoding, should be performed based on a conservedshort standardized genomic region that will enableproduction of a sufficiently divergent and broad-rang-ed primer13,14. Therefore, the DNA chip barcoding genereacts very sensitively and precisely to the target spe-cies. Various genes can be used as barcoding genes toseparate species. Species-level markers are available,including COI, cytochrome b, 16S-rDNA, ITS, and18S-rDNA as a nuclear gene, and the large subunit ofribulose 1,5-bisphosphate carboxylase/oxygenase(rbcL) as a plastid gene. In particular, the COI gene iswidely used in animals15,16. Therefore, we used theCOI gene as the barcoding gene in this study, and wewere able to identify 25 microalgal species.

Conclusions

Twenty-five different microalgal species were identi-fied from the coastal area of the South Sea in Koreausing DNA chip technology. The mitochondrial COIgene was used to identify the similarities and differ-ences in the genes for those 25 different microalgalspecies and to construct a phylogenetic tree. Additio-nally, a species-specific probe was designed at a leng-th of 21-23 bp and was used to produce the DNA chip.The microalgal chip enabled species-spot specific ex-pression using fluorescence measurements.

Materials and Methods

Marine microalgae

Marine microalgal species (Achnanthes longipes, Am-phora sp., Asterionella glacialis, Chaetoceros atlanti-cus, Chaetoceros didymus, Chaetoceros septentriona-lis, Chaetoceros vistulae, Chlorella ellipsoidea, Chlo-rophyta UF, Coscinodiscus perforatus, Cylindrothecaclosterium, Cylindrotheca fusiformis, Cymatosira lo-renziana, Ditylum brightwellii, Gloeocystis gigas,Gyrodimium impudicum, Heterosigma akashiwo, Mel-osira nummuloides, Navicula sp., Nitzschia pungens,Nitzschia subpacifica, Prorocentrum minimum, Skele-tonema costatum, Stephanopyxis turris, Thalassiosiraallenii) parceled from the Korea Marine Microalgae

330 BioChip J. (2012) 6(4): 325-334

Figure 3. Layout of DNA chip for microalgal species identi-fication. a. An assumptive image of selected probes. b. Micro-array layout for 25 probes of marine microalgal species (3-84)with position markers (1, 2). These probes were spotted on sil-yated slides in duplicate, and 4 blocks were located on themicroarray slides.

(a)

(b)

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Figure 4. DNA chip analysis using species-specific probes I. Signal intensity plot of each species. Black bars shows signalintensity. Lane 1: Achnanthes longipes, Lane 2,3: Amphora sp., Lane 4,5: Asterionella glacialis, Lane 6,7: Chaetoceros atlanticus,Lane 8,9: Chaetoceros didymus, Lane 10: Chaetoceros septentrionalis, Lane 11,12: Chaetoceros vistulae, Lane 13,14: Chlorellaellipsoidea, Lane 15,16: Chlorophyta UF, Lane 17,18: Coscinodiscus perforatus, Lane 19,20: Cylindrotheca closterium, Lane21: Cylindrotheca fusiformis, Lane 22,23: Cymatosira lorenziana, Lane 24: Ditylum brightwellii, Lane 25: Gloeocystis gigas, Lane26: Gyrodimium impudicum, Lane 27: Heterosigma akashiwo, Lane 28-30: Melosira nummuloides, Lane 31,32: Navicula sp.,Lane 33,34: Nitzschia pungens, Lane 35: Nitzschia subpacifica, Lane 36: Prorocentrum minimum, Lane 37,38: Skeletonemacostatum, Lane 39: Stephanopyxis turris, Lane 40,41: Thalassiosira allenii.

Achnanthes longipes Amphora sp. Asterionella glacialis Chaetoceros atlanticus

Chaetoceros didymus Chaetoceros septentrionalis Chaetoceros vistulae Chlorella ellipsoidea

Chlorophyta UF Coscinodiscus perforatus Cylindrotheca closterium Cylindrotheca fusiformis

Cymatosira lorenziana Ditylum brightwellii Gloeocystis gigas Gyrodimium impudicum

Heterosigma akashiwo Melosira nummuloides Navicula sp. Nitzschia pungens

Nitzschia subpacifica Prorocentrum minimum Skeletonema costatum Stephanopyxis turris

Thalassiosira allenii

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Figure 5. DNA chip analysis using species-specific probes II. Hybridization patterns for each marine microalgal species,including 25 species samples, showed species-specific signal patterns of the probes. All species were correctly identified.

Achnanthes longipes Amphora sp. Asterionella glacialis Chaetoceros atlanticus

Chaetoceros didymus Chaetoceros septentrionalis Chaetoceros vistulae Chlorella ellipsoidea

Chlorophyta UF Coscinodiscus perforatus Cylindrotheca closterium Cylindrotheca fusiformis

Cymatosira lorenziana Ditylum brightwellii Gloeocystis gigas Gyrodimium impudicum

Heterosigma akashiwo Melosira nummuloides Navicula sp. Nitzschia pungens

Nitzschia subpacifica Prorocentrum minimum Skeletonema costatum Stephanopyxis turris

Thalassiosira allenii

Culture Center (KMMCC) of Pukyong National Uni-versity and from the Korea Ocean Research & Deve-lopment Institute.

Genomic DNA preparation and DNA amplificationfor sequencing

Genomic DNA of microalgae was prepared with theG-spinGenomic DNA Extraction kit (Intron, Seoul,Korea). PCR amplifications were conducted in 20 μLreaction mixtures containing 2 μL10× reaction buf-fer, 2 μL 2.5 mM dNTPs, 0.5 μL Taq DNA polymerase(RexGene Biotech Co., Seoul, South Korea) and 2 μgtotal DNA. The COI forward primer (LCO1490), 5′-GGTCAACAAATCATAAAGATGTTGG-3′, and re-verse primer (HCO2198) 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ were used17. The PCR programwas initiated by one cycle of 95�C for 5 min, 49�C for1 min, and 72�C for 1 min, which was followed by 34cycles of 94�C for 40 sec, 49�C for 40 sec, 72�C for 1min, and a final cycle at 72�C for 5 min. MitochondrialCOI gene products were fractionated by electrophore-sis, cloned into pGEMT-Easy Vector (Promega, Madi-son, WI, USA) and used to transform E. coli DH5αcells. The microalgal COI genes were sequenced andanalyzed.

Sequence and phylogenetic analysis

Multiple sequence alignments were analyzed usingClustalW in MEGA4.0. The resulting sequence align-ment was exported to the Nexus format (Figure 1).The aligned sequences were analyzed via the distancemethod using the MEGA 4.0 program. Sequenceswere analyzed by neighbor joining in MEGA 4.018 togenerate the phylogenetic tree (Figure 2).

Probe selection

Our program selected species-specific probes withvariations of more than 2 bp in 21-23 bp. Specific pro-bes for species identification were determined usingthe results of the sequence alignment of the 25 micro-algal species. Each of the probes were 24 bases in leng-th and melting temperatures were 50-60�C19. In total,41 probes were selected from each of the 25 speciesand spotted (Table 2 and Figure 3).

Fabrication of microarray

Specific probes were selected from sequence align-ments of the mitochondrial COI genes to distinguishthe marine microalgal species. The specific probes weresynthesized by Bioneer. The species-specific probes (5μL) were mixed with 5 μL spotting buffer (6X SSCand 3 M betain). Microalgal specific probes were print-

ed onto silylated slides (Cell Associates) using a robo-tic microarrayer (Cartesian Technologies, Durham, NC,USA) and incubated at room temperature for 12-16 hr.Each slide contained six sub-arrays, allowing for paral-lel testing of four different samples.

Target gene (COI) amplification for the hybridizationexperiment

To amplify the mitochondrial COI genes, The PCRreaction was conducted in a total volume of 20 μLcontaining 10 μL of a 2× Pre-Mixed Taq polymerase(Enzynomics, Seoul, South Korea, nTaq : 2.5 units/μL, 4 mM Mg2++ (final), dNTP: 0.4 mM each (final),Glycero), 1 μL of COI specific primers (Bioneer) andDNA. The COI forward primer 5′-GGTCAACAAATCATAAAG ATGTTGG-3′, and reverse primer 5′-TAAACTTCAGGGTGACCAAAAAATCA-3′ wereused17. These were modified with CY3 fluorescent dyeto detect DNA variations by the DNA chip. PCR amp-lification was conducted in a DNA-ENGENE instru-ment (MJ Research, Waltham, MA, USA) under thefollowing conditions: preheating at 94�C for 5 min,followed by 40 cycles of 94�C for 30 sec, 49�C for 30sec, 72�C for 1 min, and a final 7 min extension at 72�C. The amplification products were electrophoresedon 1.0% agarose gel to confirm the correct size of thePCR products.

Hybridization and washing

We needed to denature the PCR product to a singlestrand, because target gene amplification was not car-ried out via an asymmetric PCR strategy. Hybridiza-tion of the amplified target sequence was conductedin 100 μL of hybridization solution (3X SSC and 0.3%sarcosyl) mixed with 10 μL of PCR product for 1 hr at55�C. The reaction solutions of the four sub-arrayswere separated from one another in a perfusion cham-ber (GraceBio, Bend, OR, USA). After hybridization,the DNA chips were immediately washed in a 1X SSCwith 0.1% sarcosyl for 5 min, 1X SSC for 5 min, and0.1X SSC for 5 min, followed by spin-drying for 5 minat room temperature.

Scanning

Hybridization signals were detected using a Genepix4000B (Axon Instrument, Sunnyvale, CA, USA) at aPMT gain of 450, and laser power of 99%. After scann-ing, the fluorescence intensity was analyzed usingGenepix 4.1 software. Image pixel intensities of eachspot were calculated to verify positive or negative re-sults, and the local background intensity was sub-tracted from the median value of each spot. Speciesdeterminations were based on the signal-to-noise

BioChip J. (2012) 6(4): 325-334 333

ratio.

Acknowledgements This work was supported by theKorea Ocean Research & Development Institute (projectNo. PE98753 and PE98785).

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