4. 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
5. Thus, authentication of herbal medicinal
materials:
- reduce unfair trade,
- raise consumers' confidence,
- ensure the therapeutic potency.
(Newmaster et al., 2013)
6. 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
7. 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
9. 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.
10.
11. • Impatiens balsamina leaf
Plant
material
• QIAGEN DNeasy Plant Mini Kit
DNA
extraction
• Nanodrop
• 1% (w/v) Agarose gel
DNA quantitative &
qualitative analysis
• rbcL, psbA-trnH, matK and
ITS2
Universal primers
design
• Gradient and single PCR
PCR
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 W2
Sequence
analysis
Methodology
13. 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
14. 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.
15. 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.
17. 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.
19. 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
20. Other types of DNA barcodes could be applied.
– ycf5,
– rpoB,
– rpoC1,
– rbcL + ITS2.
FUTURE WORK
21. 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.
22. Primer
name
Primer sequence Tm
(°C)
GC
(%)
Length
(bp)
Set1
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
Set2
matK – F
(Forward)
5' - CGT ACT TTT ATG TTT ACA GGC TAA - 3' 51 33 24
matK – R
(Reverse)
5' - TAAACG ATC CTC TCA TTC ACG A - 3' 51 41 22
Set3
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
Set4
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
23.
24. 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
25. 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).
26. 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.
27. 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).
28. 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.
29. 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).
30. 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.
31. 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).
32.
33. 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.
34. 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.
