Recombinant DNA Technology Basic Principles of Recombinant DNA Genetic manipulation techniques Genetic Modified Organisms

Recombinant DNA Technology

Basic Principles of Recombinant DNA Genetic manipulation techniques

Genetic Modified Organisms

Siti Nur Jannah

Introduction

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Biotechnology may be defined as “the method by which a living organism or its parts are used to change or to

incorporate a particular character to another living organism”

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Genetic recombination is the exchange of information between two DNA segments.

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This is a common occurrence within the same species.

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But by artificial means, when a gene of one species in

transferred to another living organism, it is called recombinant DNA technology.

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In common, this is known as genetic engineering.

Definition of recombinant DNA

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The use of technology to manipulate genes is called genetic engineering or recombinant DNA technology (rDNA technology).

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rDNA technology is a field of molecular biology in which scientists manipulate DNA to form new synthetic

molecules, called chimeras.

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Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other

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DNA fragments are usually derived from different biological sources

Definition of recombinant DNA technology

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A series of procedures used to recombine DNA segments.

Under certain conditions, a recombinant DNA molecule can enter a cell and replicate.

Basic principle of recombinant DNA technology

• The DNA is inserted into another DNA molecule called ‘vector’

• The recombinant vector is then introduced

into a host cell where it replicates itself, the

gene is then produced

Several key players:

• 1. restriction enzymes. Cut DNA at specific sequences. e.g. EcoR1 cuts at GAATTC and BamH1 cuts at GGATCC. Used by bacteria to destroy invading DNA: their own DNA has been modified

(methylated) at the corresponding sequences by a methylase.

• 2. Plasmids: independently replicating DNA circles (only circles replicate in bacteria). Foreign DNA can be inserted into a plasmid and replicated. Plasmids for cloning carry drug resistance genes that are used for selection. –Spread antibiotic resistance genes between bacterial species

• 3. DNA ligase. Attaches 2 pieces of DNA together.

• 4. transformation: DNA manipulated in vitro can be put back into the living cells by a simple process . –The transformed DNA

replicates and expresses its genes.

• Step 1: rDNA technology begins with the isolation of the gene of interest (foreign DNA). The gene is then inserted into a vector and cloned. A vector is a piece of DNA that is capable of independent growth. The commonly used vectors are bacterial plasmids and viral phages.

• The gene of interest is integrated into the plasmid (A plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. They are most commonly found in bacteria as small, circular, double-stranded DNA molecules

Schematic Diagram of Molecular Cloning

Perbanyakan DNA dengan PCR

(Polymerase Chain Reaction)

Denature (95 C)

3 Min 1 Min

1 Min

1 Min 10 Min

Annealing (60 C)

Extention (72 C)

Fig . Conditions for PCR

1. Takara LA Taq (5units/μl) 0.5μl 2. 2 x GC buffer I/II 25μl 3. DNTP Mixture (2.5 mM) 8 μl 4. DNA Template (0.5μg) 5 μl 5. Primer 1 (10 pmol) 1 μl 6. Primer 2 (10 pmol) 1 μl 7. Milli Q up to total volume 50μl

PCR Reaction ^{4 C ~}

Memotong DNA

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Menggunakan enzim endonuklease restriksi

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Ujung “lengket” (sticky ends)

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Ujung “tumpul” (blunt ends)

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Penamaan enzim

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EcoRI

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E = genus (Escherichia)

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co = species (coli)

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R = strain

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I = # of enzyme

Ujung “lengket” dan “tumpul”

(Blunt & Sticky ends)

Penyambungan (pasting) DNA

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Pembentukan ikatan- H pada ujung-ujung yang komplemen

(sticky ends)

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Ligase membentuk ikatan fosfodiester untuk merekatkan benang-benang DNA

Vektor untuk Mengklon

Diperlukan suatu wahana ( vehicle )

untuk memasukkan suatu potongan

DNA ke dalam sel agar DNA tersebut

dapat disimpan dan diperbanyak di

dalam sel tersebut

Karakteristik vektor yang baik

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Stabil

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Dapat bereplikasi sendiri

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Kecil

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Mudah diisolasi

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Mempunyai berbagai situs pemotongan tunggal

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Mudah dideteksi.

Plasmid

DNA bukan kromosom

(extrachromosomal DNA) yang secara alami dimiliki suatu jasad

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Bentuknya benang ganda (

double strands DNA, dsDNA

) sirkular

Plasmid buatan (

Artificial plasmids

) dapat dibuat

dengan menambahkan potongan-potongan DNA lain

Vektor untuk Mengklon

Plasmid dapat dimodifikasi untuk mampu membawa potongan DNA lain ke dalam sel bila memiliki:

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Replikator (origin of replication)

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^{Penanda (}

Marker

) yang mudah diseleksi

(misalnya gen ketahanan terhadap antibiotik)

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Situs untuk mengklon (potongan DNA yang memiliki urutan basa nukleotida yang menjadi sasaran enzim restriksi tetapi tidak terletak di dalam daerah replikator atau penanda

Plasmid yang Dimiliki oleh

Escherichia coli

Berasal dari plasmid alami E. coli Potongan DNA tambahan

Potongan DNA tambahan

Kloning Terorientasi

Bila diinginkan untuk menginsersikan potongan DNA asing dengan orientasi tertentu

• Dilakukan dengan memotong DNA vektor maupun DNA sumber gen yang dikehendaki menggunakan dua enzim restriksi yang

berbeda

Vektor untuk Mengklon

1 Vektor berupa plasmid

2 Vektor berupa bakteriofaga 3 Cosmid

4 BACs (Bacterial Artificial Chromosome)

& YAC (Yeast Artificial Chromosome)

1. Memiliki origin of replication dari inang yang dituju, sehingga memungkinkan replikasi secara independen terhadap genom inang.

2. Memiliki penanda selektif: Memudahkan seleksi sel pembawa plasmid tersisipi DNA asing

ketahanan terhadap antibiotik ganda penapisan biru-putih

3. Memiliki banyak situs pengkloningan (multiple cloning sites, MCS)

4. Mudah diisolasi dari sel inang.

Vektor berupa Plasmid

Vektor berupa Plasmid

Vektor berupa Plasmid

• Keunggulan:

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Kecil, mudah pengerjaannya

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Strategi seleksi mudah

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Berguna untuk mengklon potongan DNA ukuran kecil (< 10kbp)

• Kelemahan:

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Kurang bermanfaat untuk mengklon potongan DNA ukuran besar (> 10kbp)

Bakteriofaga ( l phage)

Vektor berupa bakteriofaga ( l vectors )

Vektor berupa Bakteriofaga

• Keunggulan:

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Bermanfaat untuk mengklon potongan DNA ukuran besar (10 - 23 kbp)

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Seleksi berdasar ukuran

• Kelemahan:

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Lebih sulit pengerjaannya

Vektor Cosmid

• Keunggulan:

• Bermanfaat untuk mengklon potongan DNA berukuran sangat besar (32 - 47 kbp)

• Seleksi berdasar ukuran

• Pengerjaan seperti plasmid

• Kelemahan:

• Tidak terlalu mudah untuk mengerjakan plasmid dengan ukuran sangat besar (~ 50 kbp)

Gabungan sifat vektor plasmid dan sifat berguna

dari situs l cos (dihilangkan pada vektor l)

Vektor Cosmid

Vektor BAC

• Replikasi dimediasi ori S dan ori E

• par A and par B

mengendalikan agar hanya terdapat satu vektor dalam sel

• Menggunakan

penanda ketahanan terhadap

Khloramfenikol

^R

Vecktor YAC

• Dapat disisipi gen asing 200 - 2000 kbp dan dimasukkan ke dalam yeast

telomere centromere telomere

ARS URA3 HIS3

replication origin

markers large inserts

BACs dan YACs

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Keunggulan:

• Dapat digunakan untuk mengklon potongan DNA dengan ukuran sangat besar (100 - 2,000 kbp)

• Penting digunakan dalam proyek penetapan urutan basa nukleotida total genom

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Kelemahan:

• Tidak mudah mengerjakan molekul DNA dengan ukuran sangat besar

BACs : Bacterial Artificial Chromosomes

YACs : Yeast Artificial Chromosomes

Memilih Vektor

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Ukuran DNA yang disisipkan

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Ukuran vektor

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Situs enzim restriksi yang tersedia

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Jumlah salinan (copy number)

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Efisiensi kloning

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Kemampuan untuk menapis DNA sisipan

Cara Mengklon DNA

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Isolasi vektor kloning (plasmid bacterial) & DNA sumber gen

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Pemotongan DNA sumber gen

& vektor kloning

menggunakan enzim restriksi yang sama

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Penyisipan potongan DNA sumber gen ke dalam vektor kloning yang telah dipotong menggunakan enzim restriksi yang sama; potongan

disambung dengan bantuan enzim DNA ligase

Cara Mengklon DNA (2)

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Vektor kloning yang telah tersisipi potongan DNA dimasukkan ke dalam sel inang (transformasi sel inang)

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Penapisan sel pengklon (dan gen yang

dimasukkan)

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Identifikasi sel pengklon pembawa gen yang

dikehendaki

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Transformation- process of introducing free DNA into bacteria

Competent cell- a cell that is capable of taking up DNA.

Electroporation- The use of an electric shock to momentarily open or disrupt cell walls.

Transformation

Memudahkan seleksi sel pembawa plasmid tersisipi DNA asing

❑ ketahanan terhadap antibiotik ganda

❑ penapisan biru-putih

Penanda selektif

Penapisan biru putih

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To perform blue-white screening after transformation, X- Gal is added along with Isopropyl β-D1-

thiogalactopyranoside (IPTG), an inducer of lacZ ω gene expression. The blue colonies contain bacteria with

functional β-galactosidase, indicating the plasmid taken up during transformation did not contain the DNA of interest.

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Isopropyl β-D1-thiogalactopyranoside (IPTG)

Dalam proses transformasi, sel kompeten yg dicampur dengan molekul DNA mengalami :

1. Sel Kompeten yang tidak kemasukan molekul DNA apapun 2. Sel kompeten kemasukan DNA yg tidak membawa gen X 3. Sel kompeten kemasukan DNA vektor yg membawa gen X

Bisa dilihat dari seluruh cawan A (media padat LBA) tumbuh semua yg membawa atau tidak membawa gen X.

Pada cawan B (media LBA yg diberi antibiotik), semua sel kompeten yang kosong akan mati. Karena hanya sel kompeten yg membawa plasmid resisten thd antibiotik yg hidup.

Dan pada cawn C (media LBA yg ditambah IPTG dan X-gal), warna putih koloni karena adanya kerusakan pada gen Lac-Z yg disisipi oleh gen X sedang warna biru membawa DNA plasmid tetapi tidak membawa gen X karena IPTG dan X-Gal bereaksi dg produk gen Lac-Z memberikan warna biru.

Penapisan Klon

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Medium pertumbuhan diberi antibiotik yang sesuai dengan sifat

ketahanan yang digunakan sebagai penanda, misalnya Kanamisin

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Bakteri di paruh cawan petri sebelah kanan

memiliki plasmid dengan penanda ketahanan

terhadap Kanamisin(Kan^r), yang di sebelah kiri tidak memilikinya

Penapisan warna koloni Biru/Putih

lacZ insert

Enzim tidak berfungsi

X-gal produk

lacZ

Enzim berfungsi

X-gal produk

Penapisan Koloni Bakteri pembawa

Plasmid Rekombinan

Hibridisasi Koloni

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Dapat dilakukan jika memiliki DNA pelacak

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Bagian dari gen yang dikehendaki

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Bagian dari gen yang mirip dari jasad lain

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Oligonukleotida sintetik

Molecular Farming

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The use of agricultural plants for the production of useful molecules for non food, feed or fiber

applications.

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Plants are already grown to produce valuable molecules, including many drugs.

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Molecular farming is different because the plants are genetically engineered (GE) to produce the

molecules we want them to.

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Agro bacterium tumefacians is a

bacterium that causes a disease known as crown gall in plants.

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Infects plants by transferring its genetic material into plant cell.

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Agrobacterium transformation is the most common technique for genetically engineered plants

AgroBacterium Transformation

Examples of GMO’s

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In 1994, the Flavr Savr tomato was introduced as the first GM food. It is supposed to be“tastier, firmer and fresher” than the average tomato.

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Golden rice – enriched rice containing beta-carotene (Vitamin A). This vitamin is not found in normal rice.

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^{Bt corn –} corn containing a chemical normally found in bacteria (Bacillus thuringiensis). This is toxic to insects, not humans.

Insects try to eat the plant and die.

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Herbicide resistant plants (roundup ready corn). These plants are immune to a certain herbicide, so they live while all the

other plants in the field are killed.

Image credit: Microsoft clipart

The Next Test Is The Field

Non-transgenics

Transgenics

Herbicide Resistance

Agrobacterium

A natural DNA delivery system

• A plant pathogen found in nature

• Hormone genes expressed and galls form at infection site

• Delivers DNA that encodes for plant hormones

• Infects many plant species

Gall on stem

Gall on leaf

• DNA incorporates into plant chromosome

Benefits #1

• Increased crop productivity

• This includes herbicide tolerance,

• pest and disease resistance

• E.g. “Roundup ready” crops, and BT corn.

• Could mean using less spray

Image credit: http://www.public-domain-photos.com/& Microsoft clipart

Benefits #2

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Cold tolerance

• plants developed to tolerate cold temperatures

• & withstand unexpected frost

• could destroy seedlings

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Drought & salinity tolerance

• currently inhospitable regions can now be cultivated

Image credit: http://www.public-domain-photos.com/

Benefits #3

• Improved nutrition

• crops like rice are a staple in developing countries

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nutritionally inadequate!

• GM "golden rice" is high in beta- carotene (vitamin A)

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Reduces eye-related problems like blindness due to malnutrition

Image credit: http://www.public-domain-photos.com/& Microsoft clipart

Challenges

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Human health risks

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introducing a gene into a plant may create a new allergen or cause an

allergic reaction in

susceptible individuals

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For example, inserting genes from a nut into another plant could be dangerous for people who are allergic to nuts

Image credit: Microsoft clipart

Challenges

• “

Superweeds”

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gene transfer to non-target species where herbicide tolerant plants crossbreed with

weeds potentially creating herbicide resistant weeds.

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Some Western Canadian farmers are calling Monsanto’s round-up ready canola a

superweed.

Image credit: Microsoft clipart