06 Strategi Kloning 1

TATAP MUKA 6

STRATEGI KLONING (1)

CLONE-STRATEGY

requires

DNA fragments

Genomics DNA mRNA PCR

DNA Fragment

for cloning

DNA Synthesis

• Mechanically• Restriction

enzyme

cDNA

Synthesised tothrough

Obtained from

Aligning DNA fragment to Vector

Method required

Biomolecular eecombinant

Inserted DNA /Vector concatameric

Blunt-end ligation Linker application Adapter application Homopolymer tailing Ligation of cohesive

end

Aligned to vector using

Resulted in

• cDNA lybrary• Genomics library

Resulted in

through

Can be applied for

Gene isolation

Produces

Resulted in

Learning Outcome (LO)

LO 54: menjelaskan tahapan kloning DNA

LO 55: menjelaskan materi awal kloning DNA

LO 56: menjelaskan sintesis cDNA

LO 57: menjelaskan kloning cDNA ke dalam plasmid

LO 58: menjelaskan penggunaan linker, adaptor, dan

homopolymer tailing

LO 59: menjelaskan kloning cDNA ke dalam bakteriofaga

Cloning strategies


Four steps of cloning

Cutting DNA strands into fragments

Alignment of DNA fragments to vector to allow recombinance

Propagation of DNA recombinant in the host cell

Identification and selection of targetted DNA

recombinant


cDNA synthesis

Cutting of Dna fragment using

restriction enzymeMechanicall

cuttingChemical synthesis

Transformation Transfection In vitro packing of DNA

Blunt end ligation

Linker application

Homopolymer tailing

Cohesive end ligation

Preparation of DNA fragment

Ligation

Inserting DNA to the host’s cell

Path ways

Initial matterial for cloning

LO 55: menjelaskan materi awal kloning

Genomic DNA mRNARepresenting whole genome

of an organismIf the genome is too big, it

might cause big problem especially if the main purpose is to isolate single gene

Has intron, controlling area and repeating sekuens.

Representing genetic information expressed by a particular cell

Not all genomic DNA is represented in an mRNA population .

Can only represent abundance of particular mRNA

Contains sequence of gene codes, due to elimination of intron along processing of mRNA

Note: The diversity of mRNAs is indicated by the number of different mRNA molecules. There is one mRNA that is present in chick oviduct cells at a very high level (100000 molecules per cell). This mRNA encodes ovalbumin, the major egg white protein. Source: After Old & Primrose (1989), Principles of Gene Manipulation, 4th edition, Blackwell. Mouse data from Young et al. (1976), Biochemistry 15, 2823–2828, copyright (1976) American Chemical Society. Chick data from Axel et al. (1976), Cell 11, 247–254, copyright (1976) Cell Press. Reproduced with permission.

LO 55: menjelaskan materi awal kloning

Sintesis cDNA


Fig. 6.2. Synthesis of cDNA. Poly(A) RNA (mRNA) is used as the starting material

short oligo(dT) primer is annealed to the poly(A) tail on the mRNA

reverse transcriptase to begin copying the mRNA

mRNA is removed

short double-stranded hairpin loop

second-strand synthesis by a DNA polymerase

double-stranded cDNA is trimmed with S1 nuclease

Producing cDNA from mRNA

The above statements might cause some problems as follows:1) Sinthesis of cDNA might probably not efficientlly

run especially when mRNA is relatively long.2) Application of S1 nuclease could relieve some

important bases at 5’ end.

The latest method to synthesis cDNA is by using an Oligo(dG)-primed second-strand cDNA synthesis (Figure 6.3).


Fig. 6.3. Oligo(dG)-primed second-strand cDNA synthesis


mRNA·cDNA hybrid

tailed with C residues using terminal transferase

An oligo(dG) primer is annealed to the C tails

hydrolyses the mRNA

double-stranded full-length cDNA molecule

Plasmid is still, being used as vector especially when isolation of preffered cDNA including small numbers of screened-clones.

Ligation of cDNA fragment to the vector usually run in either of the following methods : Ligation of blunt-

end linker homopolimer

tailing

Cloning of cDNA to the plasmid vector

LO 57: menjelaskan kloning cDNA ke dalam plasmid

Sinthesys of cDNA

Blunt-end ligation

linker Homopolymer tailing

Blunt-end ligation is a ligation process of DNA molecule to the blunt-end, done by which is done by DNA ligase

The blunt-end might be obtained from : Applicatiion of S1 nuclease Filling the cohesive-end with the DNA polimerase.

Blunt-ent ligation is inefficent ligation process since there is no particular relation between molecules to hold the DNA strand before the DNA ligase forms fosfodiester bonds to produce DNA recombinant.

Probability for joining two blunt-ends, the ligation might be done using high concentration DNA

Theoritically, when DNA vector is mixed with cDNA, it might produced: Recombinant with one insert. Self-ligate to make a circular molecule or insert / vector DNA could form concatemers

Practically, to avoid self ligation vector is treated by fosfatase (either BAP or CIP).

To solve problems in joining blunt-ends the following methods are applied : Linker Adaptor Homopolymer tailing

Fig. 6.4. Use of linkers. (a) The 10-mer 5’-CCGAATTCGG-3’ contains the recognition site for EcoRI. (b) The linker is added to blunt-ended DNA using DNA ligase. (c) The construct is then digested with EcoRI, which cleaves the linker to generate protruding 5’ termini.

LO 58: menjelaskan penggunaan linker, adaptor, dan homopolymer tailing

Fig. 6.5. Use of adaptors. In this example a BamHI adaptor (5’-GATCCCCGGG-3’) is annealed with a single-stranded HpaII linker (3’-GGGCCC-5’) to generate a double stranded cohesive-ended molecule, as shown in (a). This is added to blunt-ended DNA using DNA ligase. The DNA therefore gains protruding 5’ termini without the need for digestion with a restriction enzyme, as shown in (b). The 5’ terminus of the adaptor can be dephosphorylated to prevent self ligation.


Fig. 6.6. Homopolymer tailing. (a) The vector is cut with PstI, which generates protruding 3-OH termini. (b) The vector is then tailed with dG residues using terminal transferase. The insert DNA is tailed with dC residues in a similar way. (c) The dC and dG tails are complementary and the insert can therefore be annealed with the vector to generate a recombinant. The PstI sites are regenerated at the ends of the insert DNA, as shown.


(a) The ds cDNA is treated with EcoRI methylase,

(b) which methylates any internal EcoRI recognition sequences.

(c) EcoRI linkers are then added to the ends of the methylated cDNA, and the linkers digested with EcoRI.

(d) The methylation prevents digestion at internal sites, and the result is a cDNA with EcoRI cohesive ends.

(e) This can be ligated into the EcoRI site of a λ vector such as λgt10.


Cloning cDNA into bacteriophage

Fig. 6.7 Cloning cDNA in λ vectors using linkers

Cloning cDNA in phage vectors using linkers


MINGGU DEPAN TATAP MUKA 7

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06 Strategi Kloning 1