kong pui san_viva_210515

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DNA BARCODING IN Impatiens balsamina (KEEMBONG) USING CHLOROPLASTIC AND NUCLEAR MARKERS KONG PUI SAN A11QB0084 Supervisor: DR. FAEZAH MOHD SALLEH

Transcript of kong pui san_viva_210515

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DNA BARCODING IN Impatiens balsamina (KEEMBONG) USING CHLOROPLASTIC AND NUCLEAR

MARKERS

KONG PUI SAN A11QB0084

Supervisor: DR. FAEZAH MOHD SALLEH

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OUTLINE

Introduction

Materials and Methods

Result and Discussion

Conclusion and Future Work

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Combination of herbs used for medical purpose.

Health maintaining source & cure diseases in worldwide. (Cooper, 2004; Firenzuoli and Gori, 2007)

However, the herbal medicine industries have been suffering

i. adulteration ii. substitution iii. Contaminant / filler (Techen et al., 2014)

Herbal medicine

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Thus, authentication of herbal medicinal materials:

- reduce unfair trade, - raise consumers' confidence, - ensure the therapeutic potency.

(Newmaster et al., 2013)

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DNA barcoding = find one or a few DNA regions that will differentiate and analyze among the majority of the world’s species

reference library. (Hebert et al., 2003)

DNA markers:i. Chloroplastic marker (rbcL, psbA-trnH,

matK,)ii. Nuclear marker (ITS2)

DNA barcoding

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Impatiens balsamina belongs to the family Balsaminaceae and is reputed to possess beneficial chemical and pharmacological properties. (Lim, 2014)

The leaves of Impatiens balsamina used to treat:i. swelling, ii. beriberi, iii. tumours, iv. superficial infections and etc.

(Yang et al., 2001)

Impatiens balsamina

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PROBLEM STATEMENT

Purity of the products.

The effectiveness of DNA markers is unknown.

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OBJECTIVES

To isolate high quality genomic DNA (gDNA) from Impatiens balsamina.

To optimize the amplification of chloroplastic and nuclear DNA barcodes via PCR.

To verify the amplified DNA barcode via bioinformatic sequence analysis.

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• Impatiens balsamina leafPlant material

• QIAGEN DNeasy Plant Mini KitDNA extraction

• Nanodrop • 1% (w/v) Agarose gel

DNA quantitative & qualitative analysis

• rbcL, psbA-trnH, matK and ITS2

Universal primers design

• Gradient and single PCRPCR optimization

Isolation of High Quality Genomic

DNA

Amplification of DNA barcode via

PCR

Verification of DNA barcode

sequence analysis

• MyTACG Bioscience Enterprise (Malaysia)

Sequencing PCR product

• BIOEDIT, BLAST, CLUSTAL W2Sequence analysis

Methodology

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RESULTS AND DISCUSSIONS

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Table 4.1: Purity and concentration of gDNA extracted from I. balsamina using QIAGEN DNeasy Plant Mini Kit on Nanodrop spectrophotometer.

Standard ratio ~1.8

Standard ratio

2.0-2.2

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Figure 4.1 Total gDNA extracted from I. balsamina using QIAGEN DNeasy Plant Mini Kit on 1% (w/v) agarose gel electrophoresis.

Lane M: 1 kb DNA ladder (Promega),

1: I. balsamina sample 1,

2: I. balsamina sample 2.

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Figure 4.2 Amplified PCR products for four different barcodes from I. balsamina on 1 % (w/v) agarose gel electrophoresis.

Lane M: 1 kb DNA ladder (Promega), 1: rbcL, 2: psbA-trnH, 3: ITS2,4: matK, 5: negative control.

rbcL, psbA-trnH and ITS2 have single intact band, while matK has no band.

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Figure 4.3 Chromatogram of psbA-trnH gene from I. balsamina (337 bp).

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Figure 4.4 Multiple sequence alignment of the partial psbA-trnH gene from I. balsamina with I. balsamina voucher USDA OPGC 706 psbA-trnH intergenic spacer (EF590706.1). Consensus regions are indicated by black colour at the bottom of the alignment.

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CONCLUSION AND FUTURE WORK

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Genomic DNA (gDNA) of Impatiens balsamina was successfully extracted using DNeasy Plant Mini Kit (Qiagen).

psbA-trnH being the best barcode for identification of Impatiens balsamina.– shorter barcode size (337bp).– 100% match.

CONCLUSION

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Other types of DNA barcodes could be applied.– ycf5, – rpoB,– rpoC1,– rbcL + ITS2.

FUTURE WORK

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REFERENCES• Cooper, E. L. (2004). Drug discovery, CAM and natural products. Evidence-Based Complementary and

Alternative Medicine, 1(3), 215. Firenzuoli, F., & Gori, L. (2007). Herbal medicine today:

clinical and research issues.Evidence-Based Complementary and Alternative Medicine, 4(S1), 37-40.

•Hebert, P. D., Cywinska, A., & Ball, S. L. (2003). Biological identifications through DNA

barcodes. Proceedings of the Royal Society of London. Series B: Biological Sciences, 270(1512), 313-321.

• Lim, T. K. (2014). Viola tricolor. In Edible Medicinal and Non Medicinal Plants (pp. 808-817).

Springer Netherlands.

•Newmaster, S. G., Grguric, M., Shanmughanandhan, D., Ramalingam, S., & Ragupathy, S. (2013). DNA

barcoding detects contamination and substitution in North American herbal products. BMC

medicine, 11(1), 222.

•Techen, N., Parveen, I., Pan, Z., & Khan, I. A. (2014). DNA barcoding of medicinal plant material for

identification. Current opinion in biotechnology, 25, 103-110.

•Yang, X., Summerhurst, D. K., Koval, S. F., Ficker, C., Smith, M. L., & Bernards, M. A. (2001). Isolation

of an antimicrobial compound from Impatiens balsamina L. using bioassay‐guided

fractionation. Phytotherapy Research,15(8), 676-680.

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  Primer name

Primer sequence Tm(°C)

GC(%)

Length(bp)

Set

1

ITS2 - F (Forward)

5’- GGG GCG GAT ATT GGC CTC CCG TGC -3’ 68.2 70.8 24

ITS2 - R (Reverse)

5’- GAC GCT TCT CCA GAC TAC AAT - 3’ 54.2 47.6 21

Set

2

matK – F(Forward)

5' - CGT ACT TTT ATG TTT ACA GGC TAA - 3' 51 33 24

matK – R (Reverse)

5' - TAA ACG ATC CTC TCA TTC ACG A - 3' 51 41 22

Set

3

rbcL - F(Forward)

5’- CTT GGC AGC ATT CCG AGT A- 3’ 60.2 53 19

rbcL - R(Reverse)

5’- TCA CAA GCA GCA GCC AGT TC- 3’ 62.4 55 20

Set 4

 

psbA-trnH -F(Forward)

5’ - GTT ATG CAT GAA CGT AAT GCT C - 3’ 58.4 41 22

  psbA-trnH -R(Reverse)

5’ - CGC GCA TGG TGG ATT CAC AAT CC - 3’ 73.5 57 23

Universal primers used in the study.

Primers

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Steps No. of Cycles Temperature (oC) Duration (min)

Initiate cycle 1 95 2

Denaturing

30

95 1

Annealing 45-55 2

Extension 72 2

Final extension 1 72 5

Hold 1 4 ∞

PCR profile of rbcL/psbA-trnH/matK/ITS2 amplification for 30 cycles.

PCR Optimization

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rbcL gradient PCR

Figure 4.2 Gradient PCR (45-55 oC) products for rbcL gene amplification using rbcL universal primer from Impatiens balsamina on 1 % (w/v) agarose gel electrophoresis. Four µL of DNA ladder was loaded and 5 µL of each sample was loaded on the gel. Lane M: 1 kb DNA ladder (Promega), 1: 45.9 oC, 2: 46.8 oC, 3: 48.1 oC, 4: 49.4 oC, 5: 50.6 oC, 6: 51.9 oC, 7: 53.2 oC (negative control).

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rbcL single PCR

Figure 4.3 Single PCR products for rbcL gene amplification using rbcL universal primer from Impatiens balsamina on 1 % (w/v) agarose gel electrophoresis. Four µL of DNA ladder was loaded and 3 µL of each sample was loaded on the gel. Lane M: 1 kb DNA ladder (Promega), 1: rbcL sample 1, 2: rbcL sample 2, 3: rbcL sample 3, 4: negative control. Optimum annealing temperature was set at 50.6 oC.

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psbA-trnH gradient PCR

Figure 4.4 Gradient PCR (45-55 oC) products for psbA-trnH gene amplification using psbA-trnH primer from Impatiens balsamina on 1 % (w/v) agarose gel electrophoresis. Four µL of DNA ladder was loaded and 7 µL of each sample was loaded on the gel. Lane M: 1 kb DNA ladder (Promega), 1: 45 oC, 2: 45.9 oC, 3: 48.1 oC, 4: 50.6 oC, 5: 51.9 oC, 6: 53.2 oC, 7: 54.7 oC (negative control).

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psbA-trnH single PCR

Figure 4.5 Single PCR products for psbA-trnH gene amplification using psbA-trnH primer from Impatiens balsamina on 1 % (w/v) agarose gel electrophoresis. Four µL of DNA ladder was loaded and 5 µL of each sample was loaded on the gel. Lane M: 1 kb DNA ladder (Promega), 1: psbA-trnH sample 1, 2: psbA-trnH sample 2, 3: psbA-trnH sample 3, 4: negative control. Optimum annealing temperature was set at 48.1 oC.

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ITS2 gradient PCR

Figure 4.6 Gradient PCR (45-55 oC) products for ITS2 gene amplification using ITS2 primer from Impatiens balsamina on 1 % (w/v) agarose gel electrophoresis. Four µL of DNA ladder was loaded and 7 µL of each sample was loaded on the gel. Lane M: 1 kb DNA ladder (Promega), 1: 45.3 oC, 2: 46.8 oC, 3: 48.1 oC, 4: 50.6 oC, 5: 51.9 oC, 6: 53.2 oC, 7: 54.1 oC (negative control).

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ITS2 single PCR

Figure 4.7 Single PCR products for ITS2 gene amplification using ITS2 primer from Impatiens balsamina on 1 % (w/v) agarose gel electrophoresis. Four µL of DNA ladder was loaded and 5 µL of each sample was loaded on the gel. Lane M: 1 kb DNA ladder (Promega), 1: ITS2 sample 1, 2: ITS2 sample 2, 3: ITS2 sample 3, 4: negative control. Optimum annealing temperature was set at 48.1 oC.

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matK gradient PCR

Figure 4.8 Gradient PCR (45-55 oC) products for matK gene amplification using matK primer from Impatiens balsamina on 1 % (w/v) agarose gel electrophoresis. Four µL of DNA ladder was loaded and 5 µL of each sample was loaded on the gel. Lane M: 1 kb DNA ladder (Promega), 1: 45.3 oC, 2: 46.8 oC, 3: 48.1 oC, 4: 50.6 oC, 5: 51.9 oC, 6: 53.2 oC, 7: 54.1 oC (negative control).

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Figure 4.13 Multiple sequence alignment of the partial rbcL gene from Impatiens balsamina with Impatiens hoehnelii chloroplast rbcL gene for ribulose-1,5-bisphosphate carboxylase large subunit (AB043515.1). Consensus regions are indicated by black colour at the bottom of the alignment.

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Figure 4.21 Multiple sequence alignment of the partial ITS2 gene from Impatiens balsamina with Impatiens walleriana isolate IMP.37 internal transcribed spacer 1, partial sequence; 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence (KF804104.1). Consensus regions are indicated by black colour at the bottom of the alignment.