Pendekatan Biologi Molekuler Untuk Kajian

Diversitas Genetik Plasma Nutfah Lokal

(Suatu contoh implementasi pada Durian lokal di
pulau Ternate)

Dr. Sundari, S.Pd, M.Pd

Prodi Pendidikan Biologi Universitas Khairun

Mei, 2020
 outline
Ruang lingkup dan Definisi Biologi
Molekuler
Prinsip kerja Biologi Molekuler
Teknik dasar Biologi Molekuler
Kelebihan dan Kekurangan
Aplikasi Biologi Molekuler
Ruang Lingkup dan Definisi Biologi Molekuler
Biologi Molekuler (Studi Biologi tingkat
molekuler), memperlajari pondasi dari proses
replikasi,transkripsi dan translasi materi
genetik
 PRINSIP KERJA

Menganalisis komponen kimiawi di tingkat

sel atau Jaringan
Memanipulasi komponen sel atau jaringan
Menganalisis struktur gen
Merubah struktur gen melalui manipulasi
DNA
 Teknik
Pemisahan : Elektroforesis
 Modifikasi: Rekombinan DNA

Figure 20.2 An overview of how bacterial plasmids are used to clone genes
 Tools
used in
Molecular Biology
 Gel electrophoresis
• The basic principle is that DNA,
RNA, and proteins can all be
separated by means of an electric
field.
• In agarose gel electrophoresis,
DNA and RNA can be separated
on the basis of size by running the
DNA through an agarose gel.
• Proteins can be separated on the
basis of size by using an SDS-
PAGE gel, or on the basis of size
and their electric charge by using
what is known as a 2D gel
electrophoresis.
Macromolecule blotting & probing
 Tekniknya Apa Saja?
Deteksi
 Blotting:
Western Blot (protein)
Southern Blot (DNA)
Northen Blot (RNA)
 In situ Hibridisasi (IHS)
Whole Mount ISH
Section ISH
• Flourecent ISH
• Chromogenic ISH
• Imunohistochemistry (IHC)

Amplifikasi
 PCR
 RT PCR
 5’ RACE
 Bacterial amplification
 Molecular markers
• Molecular marker are based on naturally
occurring polymorphism in DNA
sequence(i.e. base pair deletion,
substitution ,addition or patterns).
• Genetic markers are sequences of DNA
which have been traced to specific
locations on the chromosomes and
associated with particular traits.
• It can be described as a variation that
can be observed.
• A genetic marker may be a short DNA
sequence, such as a sequence
surrounding a single base-pair change
(single nucleotide polymorphism, SNP), or
a long one, like mini satellites.
 Some commonly used types of genetic
markers are
• RFLP (or Restriction fragment length polymorphism)
• AFLP (or Amplified fragment length polymorphism)
• RAPD (or Random amplification of polymorphic DNA)
• VNTR (or Variable number tandem repeat)
• Micro satellite polymorphism, SSR (or Simple sequence
repeat)
• SNP (or Single nucleotide polymorphism)
• STR (or Short tandem repeat)
• SFP (or Single feature polymorphism)
• DArT (or Diversity Arrays Technology)
• RAD markers (or Restriction site associated DNA
markers)
There are 5 conditions that characterize a
suitable molecular marker
• Must be polymorphic
• Co-dominant inheritance
• Randomly and frequently distributed
throughout the genome
• Easy and cheap to detect
• Reproducible
 Molecular markers can be used for
several different applications including
• Germplasm characterization,
• Genetic diagnostics,
• Characterization of transformants,
• Study of genome
• Organization and phylogenic analysis.
• Paternity testing and the investigation of crimes.
• Measure the genomic response to selection in
livestock
RFLP (Restriction fragment length polymorphism)

RFLPs involves fragmenting a sample of DNA by a restriction enzyme,

which can recognize and cut DNA wherever a specific short
sequence occurs. A RFLP occurs when the length of a detected
fragment varies between individuals and can be used in genetic
analysis.

Advantages:
• Variant are co dominant
• Measure variation at the level of DNA sequence, not protein
sequence.
Disadvantage:
• Requires relatively large amount of DNA
 RAPD ( Random amplification of polymorphic DNA)

Random Amplification of Polymorphic DNA. It is a type of PCR

reaction, but the segments of DNA that are amplified are
random.
Advantages:
• Fast
• Relatively inexpensive
• Highly variable
Disadvantage:
• Markers are dominant
• Presence of a band could mean the individual is either
homozygous or heterozygous for the Sequence - can’t tell
which?
• Data analysis more complicated
Micro satellite polymorphism, SSR or Simple
sequence repeat
Microsatellites, Simple Sequence Repeats (SSRs), or
Short Tandem Repeats (STRs), are repeating
sequences of 1-6 base pairs of DNA.
Advantages:
• Highly variable
• Fast evolving
• Co dominant
Disadvantage:
• Relatively expensive and time consuming to develop
 • Ketelitian dan ketepatan yang tinggi
• Tidak memerlukan banyak orang
Kelebihan • Tidak memrlukan banyak ruang dan
waktu

• .
• Biaya tinggi: Alat dan bahan
• Memerlukan keahlian tertentu secara
Kelemahan ilmu dan keterampilan
Suatu contoh Riset implementasi
aplikasi DNA barcode dan RAPD
pada durian lokal di Maluku Utara
 Deskripsi Morfologi & Analisis Fenetik Durian Lokal Maluku
Utara
Variasi Morfologi Durian Lokal di Maluku
Utara

kalik kalik
ovate ovate

Pointed Pointed Canonic

concave convex al

oblong Irreguller Spreadin Intermedie

g t

Elip Ovate Oblong Obovate

shperoid biji mati

Globose Oblon Ovoi Elip Putih Krem Kuning Elip Oblong

g d
 Tahap Isolasi DNA
40 mg vorteks

Dihaluskan dengan
600 μl Nuclei Lysis 3 μl Rnase solution & Inkubasi waterbath
nitrogen cair Inkubasi waterbath
Solution dicampur 3x 37°C selama 15’
65°C selama 15’

Mix
gently Vorteks dengan Diamkan sampel
kecepatan tinggi dalam suhu ruang
selama 20’’ selama 5’

Ambil 200 μl Protein

600 μl isopropanol Sentrifugasi selama 3’
supernatan Precipitation Solution
suhu ruang kec. 13.000-16.000 x g

Tabung dibalik pada Pellet dikering

kertas tissue bersih anginkan
selama 15’
Supernatan dituang
hati-hati
600 μl etanol 70% & 100 μl DNA
Sentrifugasi selama 1’
dibolak balik Sentrifugasi selama 1’ Rehydration
kec. 13.000-16.000 x g,
kec. 13.000-16.000 x g, Solution
suhu ruang
suhu ruang

Tabung dibolak-balik secara periodik/

inkubasi semalam suhu ruang (4°C)
Inkubasi suhu 65°C
Simpan suhu 2-8°C
selama 1 jam

Promega (modifikasi), 2010

Uji Kualitatif

1 % hasil isolasi
1,5 % hasil PCR

Nanodrop
Kemurnian DNA=A260/A280

[DNA] = A260 x 50 x fp
 Amplifikasi matK

No. Sekuens primer matK (5’-3’)

1 F : ’ 5’ ATATCCGCTTATATTTCAGGAGT 3’

2 R :’ 5’GAACTAGTCGGATGGAGTAG 3’

Tahap Temperatur Waktu Siklus

(oC)
Predenaturasi 94 2’ 35x Gallaher, 2015
Denaturasi 94 2’
Annealing 62 30’’
Ekstensi 72 30’’
Post ekstensi 72 4’
 AMPLIFIKASI- RAPD
No. Primer Sekuens primer G+C (%)
(5’-3’)
1 OPA-1 CAGGCCCTTC 70%
2 OPA-2 TGCCGAGCTG 70%
3 OPA-7 GAAACGGGTG 60%
4 OPA-18 AGGTGAACCGT 60%
5 OPA-19 CAAACGTCGG 60%

Tahap Temperatur Waktu Siklus

(oC)
Predenatura 94 3’ 1x
si
Denaturasi 94 30’’ 45 x
Annealing 37 30’’
Ekstensi 72 1’30’’
(Ruwaida dkk., 2009). Post 72 7’ 1x
ekstensi
Variasi Genetik Durian Lokal berdasarkan Sekuen DNA
matK
Contoh aplikasi pada hewan
Terima Kasih