QUALITY CONTROL

DNA FINGERPRINTING

Minggu ke-11, Dr. Oeke Yunita, S.Si., M.Si., Apt., Fakultas Farmasi Universitas Surabaya
 Contoh DAFTAR TANAMAN
YANG MENGGANTIKAN TANAMAN SESUNGGUHNYA
Journal of Medicinally Active Plants, Vol 4, Issue 1, June 2015
 QUALITY ASSURANCE KETEPATAN
PRODUK HERBAL IDENTIFIKASI

•sampel dalam jumlah

IDENTIFIKASI LANGSUNG kecil ??
• Organoleptis: bau, rasa, warna dll •sampel dalam bentuk
• Morfologi: bentuk tepi daun, jenis buah dll
serbuk atau sudah
diproses ??
•sampel tanaman yang
IDENTIFIKASI MIKROSKOPIS tidak mudah diidentifikasi
??
•sampel yang seringkali
digantikan dengan sampel
IDENTIFIKASI KANDUNGAN KIMIA tanaman lain ??

IDENTIFIKASI MOLEKULER (DNA)

AUTENTIKASI
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A sequence of DNA or protein that can be screened to reveal
key attributes of its state or composition and thus used to
reveal genetic variation

Mutation
Mutation arises DNA
genetic variation at markers
the DNA level

Subsequently,
mutation arises Protein
genetic variation at
markers
DNA will cause
variation at the protein
level
TYPES OF MOLECULAR MARKERS

Biochemical
marker
Allozyme
Traditional marker
systems
Non-PCR
based marker
RFLP, Minisatellite (VNTR)

PCR based
marker PCR generation:
Microsatellite, RAPD, AFLP, CAPS in vitro DNA
(PCR-RFLP), ISSR, SSCP, SCAR, amplification
SNP, etc.
 DNA FINGERPRINTING dikenal juga sebagai
DNA profilling, genetic fingerprinting, dan
DNA typing

DNA FINGERPRINTING adalah teknik analisis

molekuler yang digunakan untuk
membedakan individu dari spesies yang
sama menggunakan marker / penanda pada
DNA

PENGEMBANG TEKNIK DNA FINGERPRINTING : Alec Jeffreys, pada 1984

(Leicester, England)

APLIKASI PLANT DNA FINGERPRINTING

• Identifikasi Kultivar
• Mempertahankan galur breeding
• KONTROL KUALITAS BAHAN BAKU PRODUK HERBAL
• KONTROL KUALITAS BAHAN HERBAL SELAMA PROSES
• IDENTIFIKASI BAHAN HERBAL DALAM PRODUK HERBAL
 SAMPEL Isolasi DNA
-Bagian tanaman
-Cairan Biologis Analisis Kualitatif (Kemurnian DNA)
-Sel/Jaringan - Spektrofotometri
(A260/A280) = 1,8-2,0
- Elektroforesis
cut by restriction enzmye
Analisis Kuantitatif (Spektrofotometri)
(A260 x faktor pengenceran x 50) ng/μl

Amplifikasi DNA
(PCR method)

Elektroforesis Sequencing
Hasil PCR
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PERTIMBANGAN PEMILIHAN
MARKER
• Genomic abundance

• Polymorphism level

• Locus specificity

• Reproducibility

• Technical requirements

• Financial investment
 • Codominance or dominace

Codominant marker: Dominant marker:

A marker in which both alleles are A marker shows dominant inheritance with
expressed, thus heterozygous individuals homozygous dominant individuals
can be distinguished from either indistinguishable from heterozygous
homozygous state individuals
 Random Amplified Polymorphic DNA (RAPD)

• RAPD is often used to show relatedness among DNA populations.

• In this procedure arbitrary (random) primers are used during PCR to
produce a fingerprint of the DNA.
• A single primer is used which must anneal in 2 places on the DNA template
and region between the primers will be amplified.
• The primers (8-10nt) are likely to anneal in
many places on the template DNA and will
produce a variety of sizes of amplified
products.
• Amplified products are separated by agarose
gel electrophoresis and visualized.
• If the samples have similar genetic make up
then the pattern of bands on the gel will be
similar and vice versa.
• This procedure is widely used to differentiate
between different cultivars/varieties of the
same plant.
• Issues to consider when using this procedure
include reproducibility, quality of DNA, and
several primers may have to be used.
 PCR – RAPD on ITS region of DNA of
S.androgynus from different locations
(Oeke Yunita & Sulisetiorini, 2013, Shaker Verlag)

OPF-07

OPF-12

OPF-15
Sauropus androgynus
katuk, star gooseberry, or
SB ST TI BI BJI BJIII PWSI PWD sweet leaf
SP M TII BJI M PWSII
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 Tools and Technology Required to
Support DNA Barcoding in Plants
• Protocols and guidelines for DNA extraction and sequencing
from herbarium specimens
• Continued improvement of PCR and sequencing protocols for
regions rich in mononucleotide repeats
• Development of DNA barcoding primers and a system to
record and predict which primers will work well in a given
taxonomic group
• Development of robust multiplex PCR protocols
• Enhancement of mini-barcodes for degraded DNAs
Xin, T. et al. (2015) Survey of Commercial Rhodiola Products
Revealed Species Diversity and Potential Safety Issues,
Scientific Reports, www.nature.com
Producing Barcode Data
Barcode data anywhere, instantly

• Data in seconds to
minutes
• Pennies per sample
• Link to reference
database
• A taxonomic GPS
• Usable by non-
specialists