BIOTECHNOLOGY

MOLECULAR TECHNIQUE FOR IDENTIFICATION

AND GENETICS ENGINER

Oleh : Muhammad Arif M. S.Pi

Review
Bioteknologi  Adalah teknologi merekayasa pertumbuhan
dan metabolisme sel hidup, baik dari mikroba,
tumbuhan, hewan maupun manusia untuk
produksi komoditi barang maupun jasa yang
bermanfaat bagi manusia.
Introduction to PCR
The Polymerase Chain Reaction

Photo courtesy of Fisher Scientific

Definition
Polymerase Chain Reaction (PCR):
A procedure to amplify a specific DNA region
 Yields millions of copies of the target region, Eksponensial
amplification.
 Makes enough DNA for further molecular work
 Is the first step in preparing DNA for :

 Sequencing
 Restriction digestion
 Bacterial cloning

Diagram by Andy Vierstraete 1999

PCR

Exponential increase of the number of copies during PCR

TEKNIK-TEKNIK MOLEKULER UNTUK DIAGNOSTIK
dan IDENTIFIKASI
Penggunaan teknik molekuler Untuk keperluan :
1.Karakterisasi dan identifikasi Mikroba:
 – Keragaman genetik
 – Hubungan filogeni/kekerabatan
2. Diagnosis: deteksi dini/cepat dari patogen/Penyakit.
3. Rekayasa Genetika, (ClONING, TRANSGENIK
ORGANISM)
Keunggulan teknik molekuler:
 • Akurasi tinggi: sensitifitas dan spesifisitas
 • Cepat
 • Dapat mendeteksi keseluruhan mikroba: Viable
but not (yet) culturable.
PCR (POLYMERASE CHAIN REACTION)
BASED METHODS KARRY MULLIS (1982)

 Teknik Molekuler berkembang setelah ditemukannya

PCR (Polymerase chain Reaction) oleh karry mullis
(1982) dimana dengan sebuah alat yang dinamakan
‘’Thermal Cycler” Proses sintesis protein/Amplifikasi
(pemanjangan) DNA dapat dilakukan secara invitro
(diluar sel).
PCR ,thermal cycler multi-block system
Tahapan teknik molekuler:
 A. Ekstraksi DNA Produk DNA Genom
 B. PCR
 1. Denaturasi, (94–96°C) pemutusan ikatan hidrogen pada
DNA Genom sehingga DNA menjadi Utas Tunggal .
2. Annealing,(45-60°C), penempelan Primer
Tm = 4 (G+C) + 2 (A+T). Annealing Temperature.
3. Ekstension (70-72°C), pemanjangan DNA Target

 C. Elektroforesis (Visualisasi )
- Identifkasi Produk PCR
1. Ekstraksi DNA
1. Ekstraksi DNA
 Video ekstraksi dna
 2. PCR

The different steps of PCR

PCR

DNA sequencing

Microarrays

Mass-spec
Roles of PCR Reagents

 Taq polymerase
 Enzyme that extends growing DNA strand complementary to
DNA template
 MgCl2
 Provides ions needed for enzyme reaction
 dNTP’s
 Nucleotides (Adenine, Cytosine, Guanine, Thymine) building
blocks for new DNA strands
 Buffer
 Maintains optimal pH for enzyme
 ddH2O
Roles of PCR Reagents

 Primers
 Anneal to single-stranded DNA template
 Provide initiation site for extension of new DNA
 Forward primer
 Anneals to DNA anti-sense strand

 Reverse primer
 Anneals to DNA sense strand

DNA template
 In this case, the product of our DNA extraction
Quick Quiz
Which of the following reagents is NOT in a master
mix?

A. MgCl2
B. Template DNA
C. ddH2O
D. dNTPs
PCR (Polymerase Chain Reaction)

 Pembuatan Premix

Bahan Jumlah (µl)

Primer:
Forward 1
Reverse 1
dNTP 2,5
10 x Buffer Ex Taq 2,5
Taq DNA Polymerase 0,2

Steril Destilation Water (SDW) 18,5

 Primer
Prod FPPos.
Pair-ID Forwad Primer Reverse Primer TmDiff.
Len. RPPos.
86
2 GATGGTCAGTGCCTCTCA CCCAGTTGTATAGCGGTA 518 2
586
Primer design
General notes on primer design in PCR
Perhaps the most critical parameter for successful PCR is the design of primers
Primer selection
Critical variables are:
- primer length
- melting temperature (Tm)
- specificity
- complementary primer sequences
- G/C content
- 3’-end sequence
Primer length
- specificity and the temperature of annealing are at
least partly dependent on primer length

- oligonucleotides between 20 and 30 (50) bases are highly sequence

specific
- primer length is proportional to annealing efficiency: in general, the longer
the primer, the more inefficient the annealing

- the primers should not be too short as specificity decreases

Contoh Desain Primer
  Sekuen Gen Hemo Forwards

 gatggatcatgaataaaactattacgttacttagtgcattattactaccactaagttttgctcacgctgcc
gagccaacattgtctccagagatggtcagtgcctctcaagtaagaagcgcgcaagcgaaacaaact
tacacttatgtccgctgctggtaccgcaccagttattcaaaagatgaacctgcgaccgattgggaatgg
gcagaaaatccagacggcagttacttcacgcttgatggctactggtggagttcggtttctttcaagaac
atgttctacacagacacaccgcaaagtgttatcaagcaacgttgtgagcaaactctggacctagcaaa
tgaaaacgctgacatcaccttctttgcagccgataaccgtttctcctacaaccatactatctggagcaac
gaccctgtcatgcagccagaccaaatcaacaaggtcgtagcattgggtgacagcttgtctgatacagg
caacatctttaatgcatcacaatggcgattcccgaatccaaatagctggttcttgggacacttctcaaac
ggttttgtgtggactgagtacattgctcaagcgaaaaacttaccgctatacaactgggctgtgggtg
gcgcggcaggcgaaaaccaatacatcgctctgactggtgtaggtgagcaagtttcctcttacttggcat
atgcgaaattagcgaaaaactacaagcctgctaataccctgtttacccttgagtttggtctaaatgactt
catgaactacaaccgtagcgtgccagaagtgaaatcagactacgcggaagccttgattaaactgacc
gatgcaggtgcgaagaacttgttgttgatgacactaccagatgcaacacgtgcaccacagtttaccta
ctcgactcaagaagaaatcaacaagatccgcgcgaagatcgtggaaatgaatgagttcatcaaagca
caagcggcgtattacactgcacaaggctacaacgttaccttgtacgatacgcatgcactgtttgaaagc
ttaacagcaaatccagagcaacacggttttgtaaacgcgagccaagcttgccaagacatcaaccgctc
ttcatcggtagattacctataccatcactcattgcgttctgagtgtgcgtcttctggctctgataagtttgt
attctgggacgtaacacacccgaccacagcaaacaccactacgtggcagaaaaaatgctagaaagta
cgaatcaattgtcaaaccatcctttctaa
Sumber ; (Yuhana et al, 2009)
Reverse
Good Primer Design
 Length (17-28bp)
 GC content 50-60%
 GC Clamp
 Tm’s between 55-80
 Avoid simple sequences – e.g. strings of G’s
 Avoid primer self complementary
 e.g. hairpins, homodimers, heterodimers
 Setting PCR Program

Tahapan Suhu Waktu

(0C)
Pre-Denaturasi 94 2 menit
Denaturasi 94 1 menit
Annealing 50 1 menit 35 siklus
Elongation 72 1 menit
Final Elongation 72 2 menit
Considerations

 Contamination can easily lead to erroneous

results
 Avoid contaminating with DNA or PCR product…

 DNA stocks, PCR reagents

 Gloves, tips, pipetters, benches

 Carefully measure reagent quantities

 Use appropriate cycling conditions
PCR Animation—3D
Pcr video 2
“ Short Video PCR”
3. Elektroforesis
  Visualisasi Produk PCR (Elektroforesis)

Gel agarosa Dipanaskan Dituang ke ke dalam

0,7% (0,21 gr sampai bening chamber yang sudah
agarose+30 ml dan diamkan dipasang sisir
TBE) hingga hangat

3 µl produk Dimasukkan ke
Dimasukkan PCR + 4 µl
1 µl marker ke sumur bak
+ 4µl loading
dalam gel elektroforesis
Loading dye/blue juice
dye
 Perendam
an dengan Diletakkan di
Running (200 atas
Volt, 70 mA) EtBr
(Ethidium ultraviolet
hingga ¾ bagian illuminator
dari lebar gel Bromida)
3’ dan
blue view Pengambilan
Dicuci gambar
aquades
DNA Sequencing

The separation of the sequencing fragments

To measure the sizes of the fragments, each of the four reactions would be loaded into a separate well on
a gel, and the fragments would be separated by gel electrophoresis
Elektroforesis
Elektroforesis

 Video elektroforesis
 The new biology lab

Laboratory Tools and Techniques

The methods used by molecular biologists to study DNA have been
developed through adaptation of the chemical reactions and biological
processes that occur naturally in cells

Many of the enzymes that copy DNA, make RNA from DNA, and
synthesize proteins from an RNA template were first characterized in
bacteria. This basic research has become fundamental to our
understanding of the function of cells and have led to immense practical
applications for studying a gene and its corresponding protein.

As science advances, so do the number of tools available that are

applicable to the study of molecular genetics.

PCR
DNA sequencing

Microarrays

Mass-spec
BIOTECH PRODUCT
Terimakasih, Semoga Bermanfaat