Recombinant DNA Technology

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• Isolation of target gene

• Selection and construction of vectors

• Ligation of target DNA and vector

• Transformation of target gene into receptor cell

• Screening for recombinant plasmids

• Expressing a cloned gene 　

Process of cloning

　Process of DNA cloning

　§1. Isolation of target gene

1. Chemical synthesis

only for simple polypeptide chain whose primary structure is clear.

2. Obtaining from genomic DNA library

3. Obtaining from cDNA library4. polymerase chain reaction (PCR)

　The genomic DNA library is a collection of the comprehensive DNA fragments representing the entire genome of a species.

　The cDNA library represents the population of mRNAs, it only contains the exons of protein’s structural genes.

mRNA

Reverse transcripase

cDNA

replication

dscDNA

vector

recombinate DNA

E. coli

recombinate DNA in E.coli

Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure (sequence). The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence.

Whereas enzymes synthesize DNA and RNA in a 5' to 3' direction, chemical oligonucleotide synthesis is carried out in the opposite, 3' to 5' direction.

Currently, the process is implemented as solid-phase synthesis using phosphoramidite method ( 固 相 亚 磷 酸 三 酯 法 ). The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized.

Products are often isolated by HPLC to obtain the desired oligonucleotides in high purity.

§1.1 Chemical synthesis of a gene

Each nucleotide addition consists of four chemical reactions: deblocking (detritylation), coupling, oxidation, and capping.

During deblocking, DMT is removed from the growing oligonucleotide resulting in a free 5’ hydroxyl group.

During coupling, an activated nucleoside phosphoramidite is joined to the newly formed 5’ hydroxyl.

During oxidation, the unstable tricoordinated phosphate triesterlinkage formed is converted to a protected tetracoordinated phosphate triester.

During capping, oligonucleotides that fail to join to an activated nucleoside are permanently blocked to prevent their further use in subsequent steps. Figure adapted from (Caruthers et al. 1987).

Oligonucleotide synthesis cycle

Step 1 - De-blocking (detritylation): The DMT group （二甲氧基三苯甲基） is removed with a solution of an acid, such as TCA ( 三氯乙酸 ) ， in an inert solvent and washed out, resulting in a free 5' hydroxyl group on the first base;

Step 2 - Coupling: A nucleoside phosphoramidite (or a mixture of several phosphoramidites 亚磷酰胺 ) is activated by an acidic azoles catalyst, tetrazole ( 四唑 ), 2-ethylthiotetrazole, 2-bezylthiotetrazole, 4,5-dicyanoimidazole, or a number of similar compounds. This mixture is brought in contact with the starting solid support (first coupling) or oligonucleotide precursor (following couplings) whose 5‘-hydroxy group reacts with the activated phosphoramidite moiety of the incoming nucleoside phosphoramidite to form a phosphite triester linkage （亚磷酸三酯键） . This reaction is very rapid and requires, on small scale, about 20 s for its completion. The phosphoramidite coupling is also highly sensitive to the presence of water and is commonly carried out in anhydrous acetonitrile （ 乙腈） . Unbound reagents and by-products are removed by washing

Oligonucleotide synthesis is carried out by a stepwise addition of nucleotide residues to the 5'-terminus of the growing chain until the desired sequence is assembled. Each addition is referred to as a synthetic cycle and consists of four chemical reactions:

Step 3 - Capping: After the completion of the coupling reaction, a small percentage of the solid support-bound 5'-OH groups (0.1 to 1%) remains unreacted and needs to be permanently blocked from further chain elongation to prevent the formation of oligonucleotides with an internal base deletion commonly referred to as (n-1) shortmers. This is done by acetylation ( 乙酰化作用 )of the unreacted 5'-hydroxy groups using a mixture of acetic anhydride and 1-methylimidazole as a catalyst. Excess reagents are removed by washing.

Step 4 - Oxidation: The newly formed tricoordinated phosphite triester linkage （亚磷酸三酯键） is not natural and is of limited stability under the conditions of oligonucleotide synthesis. The treatment of the support-bound material with iodine and water in the presence of a weak base (pyridine, lutidine, or collidine) oxidizes the phosphite triester into a tetracoordinated phosphate triester ( 磷酸三酯键 ), a protected precursor of the naturally occurring phosphate diester internucleosidic linkage.

Synthetic cycle for preparation of oligonucleotides by phosphoramidite method

(Left) Polymerase Cycling Assembly (PCA) synthesis procedure. Oligonucleotides with complementary regions are pooled and repeatedannealing and polymerase extension cycles assembly and amplify a full-length product.

(Right) Ligase Cycling Assembly (LCA) synthesis procedure. Using a thermostable ligase, repeated annealing and ligation cycles join oligonucleotides into increasingly larger strands.A final PCR step is often employed to amplify the full-length target from the incomplete products.

Overview of PCA and LCA.

Normally, at the end of each procedure, 30-50 oligonucleotides are synthesized. They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.

　1.2 Polymerase Chain Reaction

The polymerase chain reaction (PCR) is a rapid and versatile in vitro method for amplifying DNA.

　PCR reaction system

• DNA template

• A pair of primers

• DNA polymerase (ex. Taq)

• dNTPs

• Mg2+-containing buffer

　Procedures of PCR

• Denaturing: the template DNA is denatured to become ssDNA from dsDNA by heating.

• Annealing: this step allows the hybridization of the primers with target DNA.

• Extension: this process is the DNA synthesis step.

ing

The first three cycles of PCR

Gene library: a collection of different DNA sequence from an organism, each of which has been cloned into a vector for ease of purification, storage and analysis.

Genomic libraries

cDNA libraries

Gene library (made from genomic DNA)

(made from cDNA- copy of mRNA)

§1.3 Isolating a gene from gene library

§1.3.1 Genomic DNA libraries

1. Purify genomic DNA ： prokaryotes or eukaryotes;

2. Fragment this DNA : physical shearing and restriction enzyme digestion;

3. Clone the fragments into vectors;4. Transfer the constructed vectors into

recipient cells;5. Culture and amplify the clones;6. Screen the clone of target.

To make a representative genomic libraries ,genomic DNA must be purified and then broken randomly into fragments that are correct in size for cloning into the chosen vector.

Purification of genomic DNA :

Prokaryotes :extracted DNA directly from cells

remove protein, lipids and other unwanted macro-molecules by protease digestion and phase extraction.

Eukaryotes :prepare cell nuclei

Break DNA into fragments randomly:

Physical shearing : pipeting, mixing or sonicaion

Restriction enzyme digestion: partial digestion is

preferred to get a greater lengths of DNA fragments.

Genomic

libraries

Sau3A: 5’-/GATC-3’, less selectivityBamH1: 5’-G/GATCC

Selection of restriction enzyme1. Ends produced (sticky or blunt) &

The cleaved ends of the vector to be cloned

2. Whether the enzyme is inhibited by DNA modifications (CpG methylation in mammals);

3. Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.

Genomic

libraries

Vectors

According to genome’s size,we can select a proper vector to construct a library .

Vectors Plasmid λ phage cosmid YAC

insert (kb) 5 23 45 1000

The most commonly chosen genomic cloning vectorsare λ replacement vectors which must be digested with restriction enzymes o produce the two λ end fragment or λ arms between which the genomic DNA will be digested

Genomic

libraries

cos cos

Long (left)arm

short (right)arm

Exogenous DNA(~20-23 kb)

λ phage vector in cloning

cos cos

Long (left)arm

short (right)arm

Exogenous DNA(~20-23 kb)

Genomic

libraries

λ replacement vector cloning

2. Packing with a mixture of the phage coat proteins and phage DNA-processing enzymes 3. Infection and

formation of plaques

Library constructed

1. Ligation

0.preparation of arm and genomic inserts

§1.3.2 cDNA libraries

I2-1 mRNA isolation, purification

I2-2 Check theRNA integrity

I2-3 Fractionate and enrich mRNA

I2-4 Synthesis of cDNA

I2-5 Treatment of cDNA ends

I2-6 Ligation to vector

cDNA libraries

1.No cDNA library was made from prokaryotic mRNA.

• Prokaryotic mRNA is very unstable

• Genomic libraries of prokaryotes are easier to make and contain all the genome sequences.

cDNA libraries

2.cDNA libraries are very useful for eukaryotic gene analysis

• Condensed protein encoded gene libraries, have much less junk sequences.

• cDNAs have no introns genes can be expressed in E. coli directly

• Are very useful to identify new genes• Tissue or cell type specific (differential

expression of genes)

cDNA libraries

cDNA libraries

mRNA isolation

• Most eukaryotic mRNAs are polyadenylated at their 3’ ends

• oligo (dT) can be bound to the poly(A) tail and used to recover the mRNA.

AAAAAAAAAAn5’ cap

cDNA libraries

I 2 cDNA libraries

1.Traditionally method was done by pass a preparation of total RNA down a column of oligo (dT)-cellulose2.More rapid procedure is to add oligo(dT) linked to magnetic beads directly to a cell lysate and ‘pulling out’ the mRNA using a strong magnet

3.Alternative route of isolating mRNA is lysing cells and then preparing mRNA-ribosome complexes on sucrose gradients

Three methods to isolate mRNA.

cDNA libraries

Make sure that the mRNA is not degraded. Methods:Translating the mRNA : use cell-free translation system as wheat germ extract or rabbit reticulocyte lysate to see if the mRNAs can be translatedAnalysis the mRNAs by gel elctrophoresis: use agarose or polyacrylamide gels

Check the mRNA integrity

cDNA libraries

Cloning the particular mRNAs

Is useful especially one is trying to clone a particular gene rather to make a complete cDNA library.

Fractionate on the gel: performed on the basis of size, mRNAs of the interested sizes are recovered from agarose gelsEnrichment: carried out by hybridizationExample: clone the hormone induced mRNAs (substrated cDNA library)

cDNA libraries

Synthesis of cDNA :

First stand synthesis: materials as reverse transcriptase ,primer( oligo(dT) or hexanucleotides) and dNTPs (Fig 1) Second strand synthesis: best way of making full-length cDNA is to employs a ribonuclease ( RNase H ) which recognises the RNA component of a DNA:RNA hybrid and cleaves the RNA at

a number of non-specific sites .(Fig2)

cDNA libraries

Fig.1 First strand synthesis

Fig.2 Second strand synthesis

Treatment of cDNA endsBlunt end ligation of large fragment is not efficient, so we have to use special acid linkers to create sticky ends for cloning.

The process :

Move protruding 3’-ends(strand-special nuclease)

Fill in missing 3’ nucleotide (klenow fragment of DNA polyI and 4 dNTPs)

Ligate the blunt-end and linkers(T4 DNA ligase)

Restriction enzyme digestion (E.coRI )

Tailing with terminal transferase or using adaptor molecules

cDNA libraries

Ligation to vector Any vectors with an E.coRI site would suitablefor cloning the cDNA.

The process :

Dephosphorylate the vector with alkalinephosphatase

Ligate vector and cDNA with T4 DNA ligase

(plasmid or λ phage vector)

cDNA libraries

1.2.3 Screening procedures

1 Screening

2 Colony and plaque hybridization

3 Expression screening

4 Hybrid arrest and release

5 Chromosome walking (repeat screening)

Screening procedures

1. Screening

The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library .

1. Using nucleic acid probe to screen the library based on hybridization with nucleic acids.

2. Analyze the protein product.

Screening procedures

Screening libraries

Hybridization to identify the interested DNA or its RNA product

1. Radiolabeled probes which is complementary to a region of the interested geneProbes: • An oligonucleotide derived from the

sequence of a protein product of the gene• A DNA fragment/oligo from a related gene

of another species 2. Blotting the DNA or RNA on a membrane 3. Hybridize the labeled probe with DNA

membrane (Southern) or RNA (Northern) membrane

Searching the genes of interest in a DNA library

Screening procedures

2 Colony and plaque hybridization

Transfer the DNA in the plaque or colony to aNylon or nitrocellulose membrane

Phage DNA bind to the membrane directly

Bacterial colonies must be lysed to release DNA on the membrane surface.

Hybridization (in a solution Containing Nucleic acid probe)

Wash to remove unhybri-dization probe and visualize

X-ray film(radio-actively labeled )

antibody or enzyme(modified nucleotide labeled

Line up the hybridizated region orrepeated hybridization

(Alkali treatment)

Screening procedures

Transfer to nitrocelluloseor nylon membrane

Denature DNA(NaOH)Bake onto membrane

Probe with 32p-labled DNA complementary to gene of interest

Expose to film

Select positive from master plate

Keep master plate

Screening by plaque hybridization

Screening procedures

Identify the protein product of an interested gene

1.Protein activity2.Western blotting using a

specific antibody

3 Expression screening

Screening procedures

Expression screening (1)

If the inserts are cloned into an expression sites, it may be expressed. Therefore, we can screen for the expressed proteins. However, this screening may miss the right clone

Example: the EcoRI site of lgt11 vector. The inserted genes have one in six change (1/6) to be in both the correct orientation (2 possibilities; ) and reading frame (three possibilities; three nucleotide code XXX).

Screening procedures

Expression screening (2)

The procedure has similarities to the plaque hybridization protocol.

‘Plaque lift’ ( taken by placing a membrane on the dish of plaque)

Immersed in a solution of the antibody

Detected by other antibodies

Repeat cycles of screening to isolate pure plaques

Antibodies can be used to screen the expression library.

Screening procedures

4 Hybrid arrest and screen

Individual cDNA clones or pools of clones can be used to hybridize to mRNA preparation

Hybrid arrest :translate the mRNA population directly, and the inhibition of translation of some products detected.

Hybrid release translation : purify the hybrids and the hybridized mRNAs released from them and translated, it identifies the protein encoded by the cDNA clone

Screening procedures

5 Chromosome walking

Definition: To clone the desired gene by repeated isolating adjacent genomic clones from the library.

to obtain overlapping genomic clones that represent progressively longer parts of a particular chromosome .

Screening procedures

Process:

1. Prepare a probe from the end insert .

2.The probe are used to re-screen the library by colony or plaque hybridization

3.Analyzed the new isolate clones and posited them relative to the starting clone. some will be overlapping.

4. Repeated the whole process using a probe from the distal end of the second clone.

Screening procedures

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Vector arm Genomic clone insert Vector arm

Prepare probe from ends of insert

Re-screen genomic library

Restriction

Restriction map new genomic clones

Prepare new probes from distal ends of least overlapping insert.Re-screen genomic library . Restriction map new genomic clones

Chromosome walking

Chromosome walking

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