PENGANTAR TEKNIK

DASAR RISET
BIOMEDIS
Dr.dr.Ismawati,M.Biomed
 CP-MK

• Mahasiswa mampu memahami konsep teknik dasar yang digunakan dalam

riset ilmu biomedis
• Mahasiswa mampu menjelaskan aplikasi teknik dasar yang digunakan dalam
riset ilmu biomedis
• Mahasiswa mampu memahami etika dan regulasi dalam riset ilmu biomedis
 deskripsi
• Mata kuliah ini memberikan konsep yang digunakan pada teknik-teknik dasar serta contoh
aplikasinya. Selain itu, mata kuliah ini juga memberikan konsep biosafety dan biosecurity pada riset
ilmu biomedis. Mata kuliah ini adalah mata kuliah wajib Program Studi Magister Ilmu Biomedis
Fakultas Kedokteran Universitas Riau.
• TIM DOSEN:
Dr. dr. Ismawati, M.Biomed
dr. Arfianti, M.Biomed., M.Sc., Ph.D
dr. Darmawi, M.Biomed
Rahmat Azhari Kemal, M.Si
dr. Ilhami Romus, Sp.PA
 Penilaian
• Penugasan/presentasi: 20%
• UTS: 30%
• Rerata Pre dan post test praktikum: 20%
• Rerara Laporan praktikum: 30%
 overview
Overview teknik dasar biomedis (pengenalan terminologi)
• Geno-transcriptomik (PCR, microarray, sekuensing)
• Proteomik (SDS-PAGE, ELISA, IHC, MS)
Riwayat perkembangan teknik diagnostik
molekuler
 PCR
• The principle of PCR is based on the repetitive cycling of three simple reactions of
amplification that include
(1) Denaturing: At 95 C template DNA double strand separates into two
single strands.
(2) Annealing: Next the temperature is reduced to 55 C and two specific oligonucleotide
primers attach to the DNA template complementarily.
(3) Extension:The temperature is then raised again but this time to
72 C, facilitating the DNA polymerase to extend the primers at the 3’ terminus of each primer
and synthesize the complementary strands along 5’ to 3’ terminus of each template DNA
using deoxynucleotides contained in media.
 PCR
• After n cycles (approx. 30), the final PCR products will have double no. copies of template DNA in theory.
The whole process just needs 2–5 h depending on the no. and types of nucleotide.
Varian:
-Multiplex PCR
-Reverse Transcription-Polymerase Chain Reaction(RT-PCR)
synthesis ofcDNA fromRNA by reverse transcription (RT) firstly, which is then followed with amplification of a
specific cDNA by PCR
-Real-Time PCR
quantitative assay for any
amplifiable DNA sequence
 microarray
-hybridization of DNA with target molecule
- for quantitative (gene expression) or qualitative (diagnostic)
- Analysis of large numbers of genes simultaneously or to genotype multiple
regions of a genome.
- Each DNA spot contains approx. picomoles (10-12 mol) of a specific DNA
sequence, known as probes (or reporters).
Aplikasi teknik molekuler
 NUCLEOTIDE SEQUENCING OF DNA

• The determination of the order or sequence of bases along a length of DNA

• Two techniques
1. based on an enzymatic method frequently termed Sanger sequencing
2. a chemical method called Maxam and Gilbert
Sanger sequencing most popular method and many commercial kits are available
• The Sanger method is simple and elegant and mimics in many ways the natural
ability of DNA polymerase to extend a growing nucleotide chain based on an
existing template.
 Immunohistochemistry (IHC)
• This technique works on the principle of antigen-antibody (Ag-Ab) interaction and
is used for the identification of cellular or tissue constituents, i.e., an Ag by using a
specific antibody (Ab).
• The binding of Ag to an Ab is confirmed either by labelled antibody itself or by
using secondary labelling methods such as fluorescent labelled secondary Ab.
• Furthermore, the Ag-Ab interaction can also be recognized by coloured
histochemical reaction under light microscope and fluorochromes using ultraviolet
light.
• proper standardization and inclusion of negative control are essential for the
reproducibility of results.
Contoh IHC
 ELISA
• used for the diagnosis of infectious agents such as viruses, and other substances in blood.
The antigen is the substance or agent to be measured.
• antigen is immobilised on to a solid phase, either the reaction vessel or a bead.
• The most commonly used solid phase is the enzyme linked immunosorbent assay (ELISA)
plate.
• use of a coating antibody which actively traps antigen to the solid phase.
• A second antibody (antibody enzyme conjugate) which is labelled with a reporter enzyme
is allowed to bind to the immobilised antigen.
• The enzyme substrate is then added to the antigen/ antibody/enzyme complex and a
reaction, usually involving a colour change
 Triple antibody sandwich ELISA
=indirect ELISA
Double antibody sandwich ELISA
Competitive ELISA
 SDS Polyacrylamide Gel (PAGE)
electrophoresis
• SDS: sodium dodecyl sulfate : menghilangkan perbedan muatan dan konformasi di antara protein negatif
• separates proteins on the basis of their shape (size), which in turn relates to their relative molecular masses.
• A series of proteins of known molecular mass (molecular weight (Mr) markers) are run on a gel on a track
adjacent to the protein of unknown molecular mass.
• The distance each marker protein moves through the gel is measured and a calibration curve of log Mr versus
distance moved is plotted.
• The distance migrated by the protein of unknown Mr is also measured, and from the graph its log Mr and
hence Mr is calculated.
• The method is suitable for proteins covering a large Mr range (10 000–300 000).
• method is easy to perform and requires very little material.
 Immunoblotting
= western blotting
Used to identify protein after electrophoresis
The sample may be: tissue homogenate, extract of cells or other biological source
The separate protein are transferred to nitrocellulose or polyvinyl membrane
The membrane is treated with protein blocking solution to prevent non specific
binding (popular: dried milk or bsa
Direct or indirect antibody
Indirect: membrane is incubated antibody solution washing secondary
antibody-enzyme conjugate - substrate
 Referensi
• Wilson K and Walker J. Principle and techniques of Biochemistry and
molecular biology. 2010. Newyork;Cambridge University Press, 7 th edition.
• Singh SK and Kumar Dhiraj. Protocol Used in Molecular Biology.2020.
Singapore: Bentham Science Publisher.
• Soewoto H et al. Biokimia Eksperimen Laboratorium.2016. Jakarta: Widya
Medika.