2012 dna rekombinan

8/10/2019 2012 DNA Rekombinan

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Recombinant DNATechnology

Dr. Diah Rachmawati, M.Si.Fakultas Biologi UGM

Definition of recombinant DNA

Production of a unique DNA molecule byjoining together two or more DNA fragmentsnot normally associated with each other

DNA fragments are usually derived fromdifferent biological sources

A series of procedures used to recombineDNA segments. Under certain conditions,

a recombinant DNA molecule can enter acell and replicate.

Definition of recombinant DNA

technology

History of recombinant DNAtechnology

Recombinant DNA technology is one of the

recent advances in biotechnology, which was

developed by two scientists named Boyer and

Cohen in 1973.

Basic principle of recombinantDNA technology

The DNA is inserted into another DNA

molecule called vector

The recombinant vector is thenintroduced into a host cell where itreplicates itself, the gene is thenproduced

Recombinant DNA Technology

Recombinant DNA technologyincludes DNA cloning, genecloning, and molecular cloning.

DNA from one organism istransferred to a bacterialplasmid for replication

Although viruses, bacterialartificial chromosomes alsomay be used for replicatingDNA, bacterial plasmid aremost commonly used in thistechnology and are calledvectors.


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Applications of RecombinantDNA Technology

Large-scale production of humanproteins by genetically engineeredbacteria.

Such as : insulin, Growth hormone,Interferons and Blood clotting factors(VIII & IX)

Teknologi DNA Rekombinan

Teknologi DNA rekombinan=kloning molekular

Sekumpulan teknik-teknikeksperimen yang memungkinkan peneliti untukmengisolasi, mengidentifikasi, memanipulasi dan melipatgandakan fragmenDNA dalambentuk murni.

- Teknologi kloning reproduksi- Teknologikloning terapi

Produk rekayasa disebut GMO (Genetically Modified Organisms) atau

organisme transgenik

Cloning

Cloning is the process of creating genetically identicalcells or organisms by using individual animals/plants.

There are three main types of cloning: DNA cloning,reproductive cloning and therapeutic cloning.

Cloned Sheep

Cloning involves making identical copies

Cloning can mean several things:

1. To make many identical copies of a DNA molecule orparticular stretch of DNA (DNA cloning and molecularcloning).

2. To replicate an entire organism (reproductive cloning)

3. To produce undifferentiated cells (stem cells) for thepurpose of studying and treating diseases (therapeuticcloning)

Figure 8-6 Molecular Biology of the Cell( Garland Science 2008)

Reproductive cloning to replicate an entire organism

Therapeutic cloning to produce undifferentiated cells (stem cells)for the purpose of studying and treating diseases

Therapeutic cloning (research cloning) is when stem

cells are extracted to grow into a piece of human tissuewhich is encouraged to grow into a human organ fortransplant.

Therapeutic cloning


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How is it done?

DNA is extracted from a humans cell. The DNA isinserted into a womans ovum and allowed to developand produce stem cells. The stem cells are removedfrom the pre-embryo and are treated to grown intowhatever organ is needed. Thus, the new organ istransplanted into the patient.

Stem cells

What are its uses?

- It is used for medical purposes, such as creating organs to transplant

into a patient in need of that organ. If replacement organs areavailable to the sick and dying people, countless numbers of lives

could be saved. Therapeutic cloning is a fast and efficient way to

repair damaged organs.

- Therapeutic cloning can be used to make insulin-secreting cells tocure for diabetes; nerve cells to cure stroke or Parkinsons disease

Newly formed embryo containing DNA from somatic cell

cell division

implant

(Diploid )

Recombinant DNA Technology

Source of DNA

Restriction Enzymes Cloning Vector

Transformation

Selection of recombinant

Source of DNA

- Genomic DNA

- DNA copy of an mRNA atau cDNA


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Sources of DNA to Clone

Genomic DNA: cut up whole genome and clone small pieces.Advantage is, you get everything. Disadvantage is, a lot of it is

junk. Two general methods:

1. randomly shear DNA into small pieces, then ligate linkers to theends: oligonucleotides that contain a useful restriction site.

2. partially digest the DNA with a restriction enzyme that has a 4 baserecognition site. These sites will appear at random every 256 (44) basepairs. Take long pieces.

cDNA: DNA copy of mRNA, made with reverse transcriptase.Advantage: you just get the expressed genes. Disadvantages:you don't get control sequences or introns, and frequencydepends on level of expression.

Figure 8-46 Molecular Biology of the Cell( Garland Science 2008)

Genomic DNA

The coding region for a gene of interest may beinterruptedby one or more intronregions, and thusthe complete coding region could be quite long.

To a first approximation, it does not matter whichtissue we use to isolate the genomic information,i.e. the genomic content is the same in all tissues.

Genomic DNA

The genomic DNA is digested bya restriction endonuclease, and allfragments cloned at random intoa plasmid vector, then the majority ofgenetic information will be includedin the mixture of bacteria.

Cultures of the bacteria, with eachcontaining only a fraction of thegenome, collectively contain all thegenes and are called a genomiclibrary.

Genomic Library : a collection ofDNA clones that covers the entire

genome.

DNA copy of an mRNA atau cDNA

Introns will be spliced out and the mRNA will contain a contiguous

coding region.

Tissue specific expression of the protein of interest may allow usto isolate appropriate mRNA at enhanced levels, i.e. in tissues

where the protein is expressed the mRNA levels are considerably

higher than the corresponding genomic levels (there are manymore molecules of mRNA than copies of the gene).

"Reverse transcription" is a mechanism whereby genetic

information contained in mRNA is converted back into a double

stranded DNA form.

mRNA is converted to cDNA by enzymatic reactions

DNA copy of an mRNA atau cDNA

Construction of cDNA library

Isolation of total cellular RNA

mRNA molecule has at its 3 end a run of adeninnucleotide residues called a poly(A) tail.

Short oligoucleotides containing 12 to18 deoxythymidines(poly dT) acts as primers for reverse transcriptase.

Reverse transcriptase can use RNA as a template tosynthesize a DNA strand.

The product of reverse transcription is RNA-DNA hybrid


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Construction of cDNA library Construction of cDNA library

Pustaka DNA / cDNA Library

1. Populasi dari cDNA yg telah disisipkan ke dalamvektor kloning dan ditransformasi ke E. coli.

2. Lebih baik dibandingkan dengan membuat pustakagenom, sebab tdk semua DNA diekspresikan

3. Pembuatannya di mulai dari: (1) isolasi RNA, (2)pencetakan ds cDNA/complementary DNA, (3) cDNAdiligasikan dg vektor, (4) vektor ditransformasikan keE. coli

Restriction Enzymes

Origin and function

Bacterial origin = enzymes that cleave foreignDNA

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 its own DNA by methylatingthose specific sequence motifs

Enzim restriksi endonuklease

Enzim Endonuklease Restriksi : memotong DNA dengan cara mengenalurutan spesifik DNA yang akan dipotong dulu, baru melakukanpemotongan di dalam sekuen pengenal tersebut dengan hasil potongansticky end(ujung lancip) atau blunt end(ujung tumpul)

Umumnya diisolasi dari bakteri, diberi nama asal bakterinya, fungsiasalnya; menghalangi DNA masuk ke dalam sel bakteri. DNA bakteriterlindungi sebab mempunyai enzim yang memodifikasi enzim restriksihingga jadi tidak berfungsi.

Sekuen pengenal 4,6,8 pasang basa

Sifatnya palindromik: jika ditarik garis sumbu ditengah sekuen pengenalakan terlihat urutan basa yang simetris.

Memotong ikatan fosfodiester sehingga menghasilkan satu ujungmempunyai gugus fosfat dan ujung lainnya gugus OH


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Restriction Enzymes

Restriction enzymes can be used to isolate a specific gene.

Once a gene has been isolated, it can be transferred bya cloning vector to an organism.

Restriction enzymes, also called restriction endonucleases,

are enzymes that cut DNA molecules in specific places

Restriction Enzyme Mechanisms:(a)Staggered cut: leaves sticky ends

(b) Blunt End

Contoh enzim restriksi

Enzim Asal mikroorganisme Recognitionsite

Tipe pemotongan

EcoRI Escherichia coli GA-A-T-T-C

C-T-T-A-AG

5Phosphateextension

BamHI Bacillus amyloliquefaciens GG-A-T-C-C

C-C-T-A-GG

5Phosphateextension

PstI Providencia stuarti C-T-G-C-AG

GA-C-G-T-C

3Hydroxylextension

PvuII Proteus vulgaris C-A-GC-T-G

G-T-CG-A-C

Blunt end

Digesti dengan enzim restriksi

Primrose, 1994

Ligasi / Penyambungan DNA

1. Dalam kloning tahap ini diperlukan untukmenyambung DNA target dg DNA plasmid vektor

2. Untuk menyambung DNA digunakan Enzim ligase,menyambungkan ikatan fosfodiester yang terputus

3. Ujung tumpul kurang efisien penyembungannyadibanding ujung lancip

Enzim yang berperan dalam manipulasi DNA

Nuklease: memotong, memendekkan, mendegradasi

a. Eksonuklease: memotong basa satu persatu dari ujung DNAb. Endonuklease: memotong ikatan fosfodiester DNA

Ligase : menyambung DNA ss dan ds yang terputus

Polimerase : membuat kopi DNA baru berdasarkan cetakanDNA/RNA

Enzim pemodifikasi: menghilangkan atau menambah gugus

kimiawi pada DNA


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Cloning vector

a DNA molecule that carries foreign DNA into a hostcell, replicates inside a bacterial (or yeast) cell andproduces many copies of itself and the foreign DNA

Choosing the 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?

Requirements of a vector to serve as a

carrier molecule

Most vectors contain a prokaryotic origin ofreplication allowing maintenance in bacterialcells.

Some vectors contain an additional eukaryoticorigin of replication allowing autonomous,episomal replication in eukaryotic cells.

Multiple unique cloning sites are often includedfor versatility and easier library construction.

Antibiotic resistance genes and/or otherselectable markers enable identification ofcells that have acquired the vector construct.

Some vectors contain inducible or tissue-specific promoters permitting controlledexpression of introduced genes in transfectedcells or transgenic animals.

Types of Cloning Vectors

Plasmid - an extrachromosomal circular DNA molecule that autonomouslyreplicates inside the bacterial cell; cloning limit: 100 to 10,000 base pairs or0.1-10 kilobases (kb)

Phage - derivatives of bacteriophage lambda; linear DNA molecules, whose

region can be replaced with foreign DNA without disrupting its life cycle;cloning limit: 8-20 kb

Cosmids - an extrachromosomal circular DNA molecule that combinesfeatures of plasmids and phage; cloning limit - 35-50 kb

Bacterial Artificial Chromosomes (BAC) - based on bacterial mini-Fplasmids. cloning limit: 75-300 kb

Yeast Artificial Chromosomes (YAC) - an artificial chromosome thatcontains telomeres, origin of replication, a yeast centromere, and aselectable marker for identification in yeast cells; cloning limit: 100-1000 kb

Plasmid

Adalah molekul DNA sirkular untai ganda yang banyak terdapat di dalamsel bakteri, di luar kromosom

Selalu membawa 1 gen, merupakan ciri penting bakteri pembawanya.Misalnya gen tahan antibiotik

Ukuran di alam 1 kb 250 kb

Ciri khasnya yaitu mempunyai situs untuk memulai replikasi sendirisehingga mampu memperbanyak diri tidak tergantung kromosom.

Guna: mengklon fragmen DNA besar, konstruksi pustaka DNA, subkloning,memanipulasi DNA, mengkonstruksi DNA.

plasmid yang sekarang digunakan untuk rekonstruksi DNA dimodifikasidari alam, sudah dikurangi atau ditambah dengan sifat tertentu untukmempermudah pekerjaan kloning

Plasmid yang digunakan untuk kloning umumnya berukuran antara 2-4 kb.


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Plasmid vector

Covalently closed, circular, double stranded DNAmolecules that occur naturally and replicateextrachromosomally in bacteria

Many confer drug resistance to bacterial strains

Origin of replication present (ORI)

Vectors typically include a selectable

marker and a cloning site

selectable markers usually are a gene for a productthat the host cell cannot make itself, such as anantibiotic resistance factor

the cloning site on a vector is engineered with manypossible sites for restriction enzyme cutting, whereforeign DNA can be inserted

Plasmid pUC19 Plasmid

Ori/origin of replication

Digunakan untuk memperbanyak diri tanpa tergantungperbanyakan kromosom inang.

marker seleksi/selectable marker

untuk proses seleksi plasmid yang membawa rekombinan,umumnya barupa gen tahan antibiotik.

situs kloning/cloning site

Tempat dimana DNA yang akan diperbanyak disisipkan;panjangnya beberapa puluh-ratusan pasang basa; terdapatbeberapa situs enzim restriksi, situs restriksinya satu satunya

ditempat itu.

A cloning vector is a carrier that is used to clone a gene and transfer itfrom one organism to another

the piece of foreign DNA inserted at a cloning site is said to be cloned,and the combined foreign DNA + vector is called recombinant DNA

Producing Recombinant DNA

The combination of DNA from two or more sources is called

recombinant DNA. Inserting a donor gene, such as the human

gene for insulin, into a cloning vector, such as a bacterialplasmid, results in a recombinant DNA molecule.


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Introducing recombinant DNA into Cells

DNA-mediated transformation

Microinjection

Electroforation

Transfection

Transformation

The uptakeuptake of free foreign DNA into the cell

a piece of DNA to be inserted into a vector.

piece of DNA with a restriction enzyme and then ligate the DNA insert intothe vector with DNA Ligase. The insert contains a selectable marker whichallows for identification of recombinant molecules.An antibiotic marker is often used so a host cell without a vector dies whenexposed to a certain antibiotic, and the host with the vector will livebecause it is resistant.

The vector is inserted into a host cell (bacteria), in a process calledtransformation. Selectable markers can be for antibiotic resistance, colorchanges, or any other characteristic which can distinguish transformedhosts from untransformed hosts.

Different vectors have different properties to make them suitable todifferent applications. Some properties can include symmetrical cloningsites, size, and high copy number.

Transformasi DNA rekombinan keE.coli

Secara alami bakteri mampu mengambil molekul DNAdari media tempat tumbuhnya

E. colidalam keadaan normal hanya mampumengambil DNA dalam jumlah terbatas.

Harus ada perlakuan fisik dan kimiawi tertentu untukmeningkatkan kemampuan mengambil sel yang telahdiperlakukan disebut sel kompeten.

Transformasi DNA terkonstruksi keE.coli

Untuk membuat sel kompeten biasanya dimasukkanlarutan garam 50mM kalsium klorid dingin.

Kemungkinan CaCl2 menyebabkan perubahan strukturdinding sel bakteri sehingga mudah menyerap DNA

Efisiensi transformasi 0.01%

Perlu DNA penanda/selectable marker berupa DNAresistensi terhadap antibiotik misalnya pUC19seleksinya dengan ampisilin.

Electroporation

Electric Shock Opens Pores in Cell Wall

Microinjection

In microinjection, the DNA is injected directly into thenucleus of the cell being transformed.

In biolistics, the host cells are bombarded with high

velocity microprojectiles, such as particles of gold ortungsten that have been coated with DNA.


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Microparticle Bombardment

Shoots projectiles of gold or tungsten coated withDNA or RNA into cells

Generally used with Eucaryotes

Basic method of immunological screening ofrecombinants

Primrose, 1994

Analyzing the status and expression oftransferred DNA

DNA status

RNA analysis

Protein: level and biological activity

DNA status

DNA status : autonomous or integrated

Southern Blotting -- DNA cut with restriction enzymes - probed withradioactive DNA.

Total DNA digest with restriction enzymes electrophoresis transfer DNA into nitrocellulose membrane hybridize with probe.

Probe is single-stranded DNA which is homolog to the DNA of interest.

Technique of Southern Blotting

Primrose, 1994

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 gelsoaked 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


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RNA analysis

How much is produced and whether it isauthentic?

Level of RNA are quantified by Northern blotting.

Northern Blot RNA - probed with radioactiveDNA or RNA.

Protein: level and biological activity

Western blotting Protein - probed with radioactiveor enzymatically - tagged antibodies.

Western blotting: transfer of electrophoresed proteinbands from polyacrilamide gel on to a membrane.

Total protein extract is separated by polyacrylamidgel electrophoresis and the protein are stained withCoomasie blue a new protein encoded by thetransferred DNA

Protein Expression

kDa M hLF NT 1 2 3 4

100

75

50

37

rhLF

kDa M hLF NT 1 2 3 4

100

75

50

37

rhLF

SDS PAGE Western Blot

Application of recombinant DNA

technology

Protein expression

DNA sequencing (gene mapping)

Restriction fragment length polymorphism (RFLP) analysis

Diagnosis of genetic diseases, mutation.

Studies of gene regulation, protein function, etc

Transgenic organism - improved food sources (goldenrice, insect resistance, herbicide resistance etc.)

DNA technology can be used to cure diseases, to treat genetic

disorders, to improve food crops, and to do many other things thatmay improve the lives of humans.

Production of Human Insulin

1) Obtaining the human insulin gene

Human insulin gene can be obtained by making acomplementary DNA (cDNA) copy of the messenger RNA(mRNA) for human insulin.

2) Joining the human insulin geneinto a plasmid vector

The bacterial plasmids and the cDNA are mixed together.The human insulin gene (cDNA) is inserted into the

plasmid through complementary base pairing at sticky

ends.


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3)Introducing the recombinantDNA plasmids into bacteria

The bacteria E.coli is used as the host cell. If E. coliand therecombinant plasmids are mixed together in a test-tube.

4)Selecting the bacteria which havetaken up the correct piece of DNA

The bacteria are spread onto nutrient agar. The agar also containssubstances such as an antibiotic which allows growth of only the

transformed bacteria.

2012 dna rekombinan

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