Pendekatan Biologi Molekuler Untuk Kajian

Diversitas Genetik Plasma Nutfah Lokal

(Suatu contoh implementasi pada Durian lokal di

pulau Ternate)

Prodi Pendidikan Biologi Universitas Khairun

Mei, 2020

Dr. Sundari, S.Pd, M.Pd

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

Figure 20.2 An overview of how bacterial plasmids are used to clone genes

Modifikasi: Rekombinan DNA

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

• Tidak memrlukan banyak ruang dan waktu

Kelebihan

• .

• Biaya tinggi: Alat dan bahan

• Memerlukan keahlian tertentu secara ilmu dan keterampilan

Kelemahan

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

1

oblong Irreguller Spreadin

g

Intermedie

t

kalik

ovate

kalik

ovate

Ovate Oblong Obovate Elip

Globose Oblon

g

Elip Ovoi

d

Pointed

concave

Pointed

convex

Canonic

al

Putih Krem Kuning

shperoid biji mati

Elip Oblong

Tahap Isolasi DNA

Promega (modifikasi), 2010

Dihaluskan dengan

nitrogen cair

40 mg

600 μl Nuclei Lysis Solution

vorteks

Inkubasi waterbath 65°C selama 15’

3 μl Rnase solution & dicampur 3x

Inkubasi waterbath 37°C selama 15’

Diamkan sampel dalam suhu ruang

selama 5’

200 μl Protein Precipitation Solution

Vorteks dengan kecepatan tinggi

selama 20’’

Sentrifugasi selama 3’ kec. 13.000-16.000 x g

Ambil supernatan

600 μl isopropanol suhu ruang

Mix gently

Sentrifugasi selama 1’ kec. 13.000-16.000 x g,

suhu ruang

Supernatan dituang hati-hati

600 μl etanol 70% & dibolak balik Sentrifugasi selama 1’

kec. 13.000-16.000 x g, suhu ruang

Tabung dibalik pada kertas tissue bersih

Pellet dikering anginkan

selama 15’

100 μl DNA Rehydration

Solution

Tabung dibolak-balik secara periodik/ inkubasi semalam suhu ruang (4°C)

Inkubasi suhu 65°C selama 1 jam

Simpan suhu 2-8°C

Uji Kualitatif

Nanodrop Kemurnian DNA=A260/A280

[DNA] = A260 x 50 x fp

1 % hasil isolasi

1,5 % hasil PCR

Amplifikasi matK

No. Sekuens primer matK (5’-3’)

1 F : ’ 5’ ATATCCGCTTATATTTCAGGAGT 3’

2 R :’ 5’GAACTAGTCGGATGGAGTAG 3’

Tahap Temperatur

(oC)

Waktu Siklus

Predenaturasi 94 2’ 35x

Denaturasi 94 2’

Annealing 62 30’’

Ekstensi 72 30’’

Post ekstensi 72 4’

Gallaher, 2015

AMPLIFIKASI- RAPD

No. Primer Sekuens primer

(5’-3’)

G+C (%)

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%

(Ruwaida dkk., 2009).

Tahap Temperatur

(oC)

Waktu Siklus

Predenatura

si

94 3’ 1x

Denaturasi 94 30’’ 45 x

Annealing 37 30’’

Ekstensi 72 1’30’’

Post

ekstensi

72 7’ 1 x

Variasi Genetik Durian Lokal berdasarkan Sekuen DNA

matK

Contoh aplikasi pada hewan

Terima Kasih