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Definition of recombinant DNA

Recombinant DNA
Technology
Dr. Diah Rachmawati, M.Si.
Fakultas Biologi UGM

Definition of recombinant DNA
technology
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 

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

DNA fragments are usually derived from
different biological sources

History of recombinant DNA
technology

Recombinant DNA technology is one of the
recent advances in biotechnology, which was
developed by two scientists named Boyer and
Cohen in 1973.

Recombinant DNA Technology 
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

includes DNA cloning, gene
cloning, and molecular cloning. 
DNA from one organism is

transferred to a bacterial
plasmid for replication 
Although viruses, bacterial

artificial chromosomes also
may be used for replicating
DNA, bacterial plasmid are
most commonly used in this
technology and are called
vectors.

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Interferons and Blood clotting factors (VIII & IX) Teknologi DNA Rekombinan Teknologi DNA rekombinan=kloning molekular Sekumpulan teknik-teknik eksperimen yang memungkinkan peneliti untuk mengisolasi. reproductive cloning and therapeutic cloning. To make many identical copies of a DNA molecule or particular stretch of DNA (DNA cloning and molecular cloning). mengidentifikasi. Growth hormone. Cloning can mean several things: There are three main types of cloning: DNA cloning. Such as : insulin. To replicate an entire organism (reproductive cloning) 3.Teknologi kloning reproduksi . 2.  Reproductive cloning → to replicate an entire organism  Therapeutic cloning → to produce undifferentiated cells (stem cells) for the purpose of studying and treating diseases Figure 8-6 Molecular Biology of the Cell (© Garland Science 2008) 2 .Applications of Recombinant DNA Technology   Large-scale production of human proteins by genetically engineered bacteria. memanipulasi dan melipatgandakan fragmen DNA dalam bentuk murni.Teknologi kloning terapi Produk rekayasa disebut GMO (Genetically Modified Organisms) atau organisme transgenik Cloning involves making identical copies Cloning Cloning is the process of creating genetically identical cells or organisms by using individual animals/plants. . 1. To produce undifferentiated cells (stem cells) for the purpose of studying and treating diseases (therapeutic cloning) Cloned Sheep Therapeutic cloning Therapeutic cloning (research cloning) is when stem cells are extracted to grow into a piece of human tissue which is encouraged to grow into a human organ for transplant.

Genomic DNA . The DNA is inserted into a woman’s ovum and allowed to develop and produce stem cells. nerve cells to cure stroke or Parkinson’s disease (Diploid ) Newly formed embryo containing DNA from somatic cell cell division implant Recombinant DNA Technology  Source of DNA Source of DNA  Restriction Enzymes  Cloning Vector  Transformation  Selection of recombinant .It is used for medical purposes. . countless numbers of lives could be saved. Thus. the new organ is transplanted into the patient. such as creating organs to transplant into a patient in need of that organ. Stem cells What are its uses? . Therapeutic cloning is a fast and efficient way to ‘repair’ damaged organs. If replacement organs are available to the sick and dying people.Therapeutic cloning can be used to make insulin-secreting cells to cure for diabetes.DNA copy of an mRNA atau cDNA 3 . The stem cells are removed from the pre-embryo and are treated to grown into whatever organ is needed.How is it done? DNA is extracted from a human’s cell.

 cDNA: DNA copy of mRNA. These sites will appear at random every 256 (44) base pairs. Disadvantages: you don't get control sequences or introns. in tissues where the protein is expressed the mRNA levels are considerably higher than the corresponding genomic levels (there are many more molecules of mRNA than copies of the gene). with each containing only a fraction of the genome.  Short oligoucleotides containing 12 to18 deoxythymidines (poly dT) acts as primers for reverse transcriptase. i. collectively contain all the genes and are called a genomic library. DNA copy of an mRNA atau cDNA DNA copy of an mRNA atau cDNA  Introns will be spliced out and the mRNA will contain a contiguous coding region. the genomic content is the same in all tissues. and frequency depends on level of expression. and all fragments cloned at random into a plasmid vector. i. The genomic DNA is digested by a restriction endonuclease. you get everything. "Reverse transcription" is a mechanism whereby genetic information contained in mRNA is converted back into a double stranded DNA form. Disadvantage is.  Isolation of total cellular RNA    mRNA molecule has at its 3’ end a run of adenin nucleotide residues called a poly(A) tail. Cultures of the bacteria.  Reverse transcriptase can use RNA as a template to mRNA is converted to cDNA by enzymatic reactions  The product of reverse transcription is RNA-DNA hybrid synthesize a DNA strand. and thus the complete coding region could be quite long. partially digest the DNA with a restriction enzyme that has a 4 base recognition site.Sources of DNA to Clone  Genomic DNA: cut up whole genome and clone small pieces.e. Genomic Library : a collection of DNA clones that covers the entire genome. Construction of cDNA library  Tissue specific expression of the protein of interest may allow us to isolate appropriate mRNA at enhanced levels. Advantage: you just get the expressed genes. 4 . Advantage is. Take long pieces.  To a first approximation. Figure 8-46 Molecular Biology of the Cell (© Garland Science 2008) Genomic DNA Genomic DNA  The coding region for a gene of interest may be interrupted by one or more intron regions.e. then the majority of genetic information will be included in the mixture of bacteria. then ligate linkers to the ends: oligonucleotides that contain a useful restriction site. 2. randomly shear DNA into small pieces.  Two general methods:   1. it does not matter which tissue we use to isolate the genomic information. a lot of it is junk. made with reverse transcriptase.

Lebih baik dibandingkan dengan membuat pustaka genom. (4) vektor ditransformasikan ke E. menghalangi DNA masuk ke dalam sel bakteri. (2) pencetakan ds cDNA/complementary DNA. coli. 2. coli Origin and function  Bacterial origin = enzymes that cleave foreign DNA  Named after the organism from which they were derived   EcoRI from Escherichia coli BamHI from Bacillus amyloliquefaciens  Protect bacteria from bacteriophage infection  Restricts viral replication  Bacterium protects it’s own DNA by methylating Restriction Enzymes Enzim restriksi endonuklease  Enzim Endonuklease Restriksi : memotong DNA dengan cara mengenal urutan spesifik DNA yang akan dipotong dulu. (3) cDNA diligasikan dg vektor.Construction of cDNA library Construction of cDNA library Pustaka DNA / cDNA Library 1.  Memotong ikatan fosfodiester sehingga menghasilkan satu ujung mempunyai gugus fosfat dan ujung lainnya gugus OH those specific sequence motifs 5 . sebab tdk semua DNA diekspresikan 3. diberi nama asal bakterinya.8 pasang basa  Sifatnya palindromik: jika ditarik garis sumbu ditengah sekuen pengenal akan terlihat urutan basa yang simetris. Pembuatannya di mulai dari: (1) isolasi RNA. DNA bakteri terlindungi sebab mempunyai enzim yang memodifikasi enzim restriksi hingga jadi tidak berfungsi.6. fungsi asalnya. baru melakukan pemotongan di dalam sekuen pengenal tersebut dengan hasil potongan sticky end (ujung lancip) atau blunt end (ujung tumpul)  Umumnya diisolasi dari bakteri. Populasi dari cDNA yg telah disisipkan ke dalam vektor kloning dan ditransformasi ke E.  Sekuen pengenal 4.

it can be transferred by a cloning vector to an organism. Once a gene has been isolated. Eksonuklease: memotong basa satu persatu dari ujung DNA b. memendekkan. menyambungkan ikatan fosfodiester yang terputus 3.Restriction Enzyme Mechanisms: Restriction Enzymes (a)Staggered cut: leaves “sticky ends” Restriction enzymes. 1994 Ligasi / Penyambungan DNA 1. Digesti dengan enzim restriksi Contoh enzim restriksi Enzim Asal mikroorganisme EcoRI Escherichia coli G↓ ↓A-A-T-T-C ↑G C-T-T-A-A↑ 5‘Phosphate extension BamHI Bacillus amyloliquefaciens G↓ ↓G-A-T-C-C C-C-T-A-G↑ ↑G 5‘Phosphate extension PstI Providencia stuarti C-T-G-C-A↓ ↓G G↑ ↑A-C-G-T-C 3‘Hydroxyl extension PvuII Proteus vulgaris C-A-G↓C-T-G G-T-C↑G-A-C Blunt end Recognition site Tipe pemotongan Primrose. mendegradasi a. Endonuklease: memotong ikatan fosfodiester DNA Ligase : menyambung DNA ss dan ds yang terputus Polimerase : membuat kopi DNA baru berdasarkan cetakan DNA/RNA Enzim pemodifikasi: menghilangkan atau menambah gugus kimiawi pada DNA 6 . Ujung tumpul kurang efisien penyembungannya dibanding ujung lancip Enzim yang berperan dalam manipulasi DNA Nuklease: memotong. Dalam kloning tahap ini diperlukan untuk menyambung DNA target dg DNA plasmid vektor 2. Untuk menyambung DNA digunakan Enzim ligase. are enzymes that cut DNA molecules in specific places (b) Blunt End Restriction enzymes can be used to isolate a specific gene. also called restriction endonucleases.

7 . replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA Requirements of a vector to serve as a carrier molecule  Most vectors contain a prokaryotic origin of replication allowing maintenance in bacterial cells.Choosing the Vector Cloning vector  Depends on the size of DNA to be cloned  Is the protein encoded by the DNA going to be expressed in a prokariotic or eukaryotic cell? a DNA molecule that carries foreign DNA into a host cell. episomal replication in eukaryotic cells.1-10 kilobases (kb)  Phage . cloning limit: 75-300 kb contains telomeres. cloning limit: 100 to 10.derivatives of bacteriophage lambda.  Multiple unique cloning sites are often included  Some vectors contain inducible or tissue- specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals.  Antibiotic resistance genes and/or other selectable markers enable identification of cells that have acquired the vector construct. memanipulasi DNA. cloning limit: 100-1000 kb sehingga mampu memperbanyak diri tidak tergantung kromosom.based on bacterial mini-F  Guna: mengklon fragmen DNA besar. konstruksi pustaka DNA. cloning limit .an extrachromosomal circular DNA molecule that autonomously replicates inside the bacterial cell. linear DNA molecules. merupakan ciri penting bakteri pembawanya.35-50 kb plasmids. sudah dikurangi atau ditambah dengan sifat tertentu untuk mempermudah pekerjaan kloning  Plasmid yang digunakan untuk kloning umumnya berukuran antara 2-4 kb. for versatility and easier library construction. a yeast centromere. Types of Cloning Vectors  Plasmid .  Some vectors contain an additional eukaryotic origin of replication allowing autonomous. origin of replication. dari alam. di luar kromosom  Selalu membawa ≥ 1 gen. whose region can be replaced with foreign DNA without disrupting its life cycle.000 base pairs or 0.an extrachromosomal circular DNA molecule that combines  Ciri khasnya yaitu mempunyai situs untuk memulai replikasi sendiri  Bacterial Artificial Chromosomes (BAC) . mengkonstruksi DNA. and a selectable marker for identification in yeast cells.  Yeast Artificial Chromosomes (YAC) . Misalnya gen tahan antibiotik  Ukuran di alam 1 kb – 250 kb  Cosmids .an artificial chromosome that  plasmid yang sekarang digunakan untuk rekonstruksi DNA dimodifikasi features of plasmids and phage. subkloning. cloning limit: 8-20 kb Plasmid  Adalah molekul DNA sirkular untai ganda yang banyak terdapat di dalam sel bakteri.

Producing Recombinant DNA A cloning vector is a carrier that is used to clone a gene and transfer it from one organism to another the piece of foreign DNA inserted at a cloning site is said to be cloned. into a cloning vector. double stranded DNA  selectable markers usually are a gene for a product molecules that occur naturally and replicate extrachromosomally in bacteria  Many confer drug resistance to bacterial strains  Origin of replication present (ORI)  the cloning site on a vector is engineered with many Plasmid pUC19 that the host cell cannot make itself. 8 .Plasmid vector Vectors typically include a selectable marker and a cloning site  Covalently closed. situs restriksinya satu satunya ditempat itu. such as an antibiotic resistance factor possible sites for restriction enzyme cutting. terdapat beberapa situs enzim restriksi. umumnya barupa gen tahan antibiotik. circular. such as the human gene for insulin. such as a bacterial plasmid. and the combined foreign DNA + vector is called recombinant DNA The combination of DNA from two or more sources is called recombinant DNA.  marker seleksi/selectable marker untuk proses seleksi plasmid yang membawa rekombinan.  situs kloning/cloning site Tempat dimana DNA yang akan diperbanyak disisipkan. results in a recombinant DNA molecule. where foreign DNA can be inserted Plasmid  Ori/origin of replication Digunakan untuk memperbanyak diri tanpa tergantung perbanyakan kromosom inang. Inserting a donor gene. panjangnya beberapa puluh-ratusan pasang basa.

 Different vectors have different properties to make them suitable to different applications. color changes. Transformasi DNA rekombinan ke E. or any other characteristic which can distinguish transformed hosts from untransformed hosts. Some properties can include symmetrical cloning sites.coli  Untuk membuat sel kompeten biasanya dimasukkan larutan garam 50mM kalsium klorid dingin. Electroporation Microinjection  In microinjection.  In biolistics. Electric Shock Opens Pores in Cell Wall 9 . size. coli dalam keadaan normal hanya mampu mengambil DNA dalam jumlah terbatas. the DNA is injected directly into the nucleus of the cell being transformed. and high copy number.  Kemungkinan CaCl2 menyebabkan perubahan struktur dinding sel bakteri sehingga mudah menyerap DNA  Efisiensi transformasi 0. such as particles of gold or tungsten that have been coated with DNA. The insert contains a selectable marker which allows for identification of recombinant molecules. An antibiotic marker is often used so a host cell without a vector dies when exposed to a certain antibiotic. Selectable markers can be for antibiotic resistance.  piece of DNA with a restriction enzyme and then ligate the DNA insert into  DNA-mediated transformation  Microinjection  Electroforation  Transfection the vector with DNA Ligase.coli  Secara alami bakteri mampu mengambil molekul DNA dari media tempat tumbuhnya  E. and the host with the vector will live because it is resistant. in a process called transformation.01%  Perlu DNA penanda/selectable marker berupa DNA resistensi terhadap antibiotik misalnya pUC19 seleksinya dengan ampisilin.Introducing recombinant DNA into Cells Transformation The uptake of free foreign DNA into the cell  a piece of DNA to be inserted into a vector. the host cells are bombarded with high velocity microprojectiles. Transformasi DNA terkonstruksi ke E.  The vector is inserted into a host cell (bacteria).  Harus ada perlakuan fisik dan kimiawi tertentu untuk meningkatkan kemampuan mengambil sel yang telah diperlakukan disebut sel kompeten.

 Protein: level and biological activity Technique of Southern Blotting Southern Blotting  Digest DNA with restriction endonuclease  Perform agarose gel electrophoresis of the DNA fragment from different digests  DNA fragments fractionated by size visible under UV light if gel soaked in ethidium bromide  Transfer (blot) gel to nitrocellulose filter using southern blot technique  DNA fragment are bounds to the filter  Hybridize filter with radioactive labeled probe  Expose filter to X-ray film resulting autoradiograph from hybridized DNA fragment Primrose.DNA cut with restriction enzymes .probed with radioactive DNA. 1994 DNA status Analyzing the status and expression of transferred DNA  DNA status : autonomous or integrated  Southern Blotting -. 1994 10 .  RNA analysis  Probe is single-stranded DNA which is homolog to the DNA of interest.  Total DNA → digest with restriction enzymes → electrophoresis →  DNA status transfer DNA into nitrocellulose membrane hybridize with probe.Basic method of immunological screening of recombinants Microparticle Bombardment Shoots projectiles of gold or tungsten coated with DNA or RNA into cells Generally used with Eucaryotes Primrose.

The human insulin gene (cDNA) is inserted into the plasmid through complementary base pairing at sticky ends. authentic?  Level of RNA are quantified by Northern blotting. Western Blot 3 rhLF 75→ 50→ 50→ 37→ 37→ rhLF Production of Human Insulin 1) Obtaining the human insulin gene Human insulin gene can be obtained by making a complementary DNA (cDNA) copy of the messenger RNA (mRNA) for human insulin. insect resistance.probed with radioactive DNA or RNA. to treat genetic disorders. to improve food crops.  Total protein extract is separated by polyacrylamid gel electrophoresis and the protein are stained with Coomasie blue → a new protein encoded by the transferred DNA Application of recombinant DNA technology Protein Expression SDS PAGE kDa M hLF NT 1 2 4 kDa M hLF NT 1 2 3 4 100→ 100→ 75→ DNA technology can be used to cure diseases. Studies of gene regulation.  Northern Blot → RNA . 11 .  Western blotting: transfer of electrophoresed protein bands from polyacrilamide gel on to a membrane. protein function.) 2) Joining the human insulin gene into a plasmid vector  The bacterial plasmids and the cDNA are mixed together. mutation. etc Transgenic organism .Protein: level and biological activity RNA analysis  Western blotting → Protein . herbicide resistance etc.probed with radioactive  How much is produced and whether it is or enzymatically .       Protein expression DNA sequencing (gene mapping) Restriction fragment length polymorphism (RFLP) analysis Diagnosis of genetic diseases.tagged antibodies.improved food sources (golden rice. and to do many other things that may improve the lives of humans.

coli is used as the host cell. If E. The agar also contains substances such as an antibiotic which allows growth of only the transformed bacteria. coli and the recombinant plasmids are mixed together in a test-tube.3)Introducing the recombinant DNA plasmids into bacteria The bacteria E. 4)Selecting the bacteria which have taken up the correct piece of DNA The bacteria are spread onto nutrient agar. 12 .