Basic Techniques of Genetic Engineering Teknik Dasar Rekayasa Genetika

Dwi HartantiSuparmanFaculty of PharmacyMuhammadiyah University of Purwokerto

Five Basic Techniques

Isolasi DNA/RNA Gel electroforesis DNA sequensing Hibridisasi (Hibridization) Polimerase Chain Reaction (PCR)

DNA Isolation Tujuan: mendapatkan DNA murni yang

siap dipergunakan untuk tujuan lain. Langkah:

1. Pengumpulan sel 2. Penambahan Lysis buffer untuk memecah

membran sel dan membran inti dan melepaskan isi inti sel.

3. Panambahan protease untuk mendegradasikan protein

4. Presipitasi DNA dengan menggunakan alkohol dingin dalam kadar garam yang tinggi.

DNA Isolation: Steps

DNA Isolation: Lysis Buffer & Adding Protease

50 mM Tris-HCI, pH 8.0 digunakan untuk menjaga pH larutan, karena DNA stabil pada pH 8

1% SDS digunakan untuk memecah membran sel dan membran inti sel, sehingga DNA akan bebas (SDS juga mendenaturasi dan membuka ikatan protein, sehingga protein yang ada menjadi lebih peka terhadap kerja protease)

Protease merusak protein inti yang mengikat DNA dan enzim sitoplasmik yang fungsinya memecah dan merusak DNA.

Penambahan protease akan meningkatkan rendemen DNA yang terekstraksi.

DNA Isolation: Adding Salt

Penambahan NaCI membuat molekul DNA berkumpul satu sama lain, sehingga akan mempermudah proses presipitasi.

Ion Na+ mengikat gugus fosfat molekul DNA, sehingga akan menetralkan muatan listrik DNA.

Biasanya protease sudah sekalian mengandung garam.

DNA Isolation: Precipitation of DNA

DNA tidak larut dalam alkohol Penambahan alkohol dingin akan membuat

DNA menggumpal dan terpresipitasi dari larutan.

Molekul DNA yang terpresipitasi terlihat seperti tali panjang yang berserabut seperti jaring laba-laba.

DNA bisa bertahan selama beberapa tahun bila disimpan dalam vial kaca

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Gel Electrophoresis:Definition

Gel Elektroforesis adalah proses pemisahan molekul DNA berdasarkan ukuran atau muatan listrik molekul

Gel Electrophoresis:Basic Principle

Gel Electrophoresis:Basic Principle

Menggunakan gel agarosa sebagai matriks.

Molekul DNA memiliki rangka yang terusun atas gula dan fosfat, dimana gugus fosfat akan memberikan muatan negatif yang amat kuat.

Gel agarosa dilatakkan pada medan listrik, dimana katoda terletak pada ujung yang berlawanan dengan lokasi peletakan sampel

DNA akan bergerak melalui gel, tertarik oleh katoda.

DNA yang berukuran kecil akan berpindah lebih jauh dibanding DNA yang ukurannya lebih besar.

Gel Electrophoresis:Basic Principle

UNtuk analisis,fragmen DNA yang telah terpisah divisualisasikan dengan pewarnaan sehingga bisa diamati.

Pewarnaan dilakukan dengan merendam gel dalam larutan Ethidium bromide (EtBr) yang akan memberikan fluorosensi orange.

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Polimerase Chain Reaction(PCR):Definition

PCR adalah suatu reaksi yang dilakukan didalam tabung reaksi dengan cara mencampur DNA dengan seperangkat is a reaction carried out in a single test tube by mixing DNA with a set of reagents and placing the tube in a thermal cycler than enables the mixture to be incubated at a series of temperatures that are varied in a preprogrammed manner

The Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA

First described in 1985, Nobel Prize for Kary Mullis in 1993 The primary materials, or reagents, used in PCR are:

DNA nucleotides, the building blocks for the new DNA Template DNA, the DNA sequence that you want to amplify Primers, single-stranded DNAs between 20 and 50 nucleotides

long (oligonucleotides) that are complementary to a short region on either side of the template DNA

DNA polymerase, a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA

Polimerase Chain Reaction (PCR):The Basic Steps

1. Target DNA strands is separated by heating at 94oC for 5 minutes

2. Add primers, nucleotides (ATP, CTP, GTP and TTP) and DNA polymerase

3. Cool to 60oC for a few minutes, during this time primer will attach to single stranded DNA and DNA polymerase will synthesize complementary strands

4. Repeat cycle of heating and cooling until enough copies of target DNA have been produced

PCR: the Number of Copies During PCR

Every cycle results in a doubling of the number of strands DNA present After the first few cycles, most of the product DNA strands made are the

same length as the distance between the primers The result is a dramatic amplification of a the DNA that exists between the

primers. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 1012

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DNA Sequensing: Methods The process of determining the order of the nucleotide bases

along a DNA strand is called DNA sequencing This method is based on the principle that single-stranded

DNA molecules that differ in length by just a single nucleotide can be separated from one another using polyacrylamide gel electrophoresis

Concept: If we know the distance of each type of base from a known origin, then it is possible to deduce the sequence of the DNA.

For example, if we knew that there was an: A at positions 2, 3, 11, 13 ... and G at positions 1, 12, ... and C at positions 6, 7, 8, 10, 15... and T at positions 4, 5, 9, 14....

Then we can reconstruct the sequence

DNA Sequensing: Steps

1. Preparation of reaction mixture

2. Synthesize DNA3. Gel

electrophoresis4. Autoradiography

to detect radioactive bands

DNA Sequencing: Preparation of Reaction Mixture & Synthesize DNA

Requiring many single stranded DNA fragments as template for synthesis must be isolated first

Purified single strand DNA fragment are distributed into four different tube, which containing a single modified base called dideoxynucleotide

To each tube is added a mix of nucleotide (A,C,T,G), DNA polymerase, and primer molecules that has been radioactively labeled

The absence of an oxygen on a specific carbon means that once a dideoxynucleotide is incorporated into DNA chain, no other nucleotide can be added DNA chain that contain a dideoxynucleotide is terminated at that point

These reactions are occurred randomly This method is called chain-terminating sequencing or dideoxy sequencing

DNA Sequencing: Preparation of Reaction Mixture & Synthesize DNA

DNA Sequensing: Gel Electrophoresis

The partial second strands must be removed from their complimentary first strands denaturation

The denaturated contents of each tube are loaded into a different lane of a gel to separate them

DNA Sequencing: Autoradiography

Dideoxynucleotide used in this method contain radioactive phosphorus every terminated DNA fragments is radioactive and can expose X ray film.

After the gel is run, X ray film is laid over it and exposed the film will develop a pattern of bands exactly like the one on gel it self

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DNA Hybridization

DNA Hybridization is based on the ability of complementary nucleic acid sequences to bind each other

It is useful for determining which colony/cells contain the inserted gene

DNA Hybridization: Steps to Determining the Right Inserted Cells

Prepare master plate with colonies of bacteria containing cloned segments of insulin genes

Hybridization:1. Make replica of master plate on nitrocellulose filter2. Treat filter with detergent to lyse bacteria3. Treat filter with NaOH to separate DNA into single strand4. Add radioactively labeled probes (single stranded DNA

with base sequences complimentary to that gene of interest

5. Probe will hybridize with desired gene from bacteria cell6. Wash filter to remove unbound probes and exposed filter

to X ray film7. Developed film is compared with replica of master plate

to identify colonies containing insulin gene

DNA Hybridization: Steps to Determining the Right Inserted Cells