Structural Biochemistry/DNA recombinant techniques/DNA synthesis

General Information
DNA can be synthesized by automated solid-phase technique, just like the synthesize polypeptides. A monomer is attached to a insoluble column, then activated monomers are added in the order of the sequence interested to form an oligonucleotide. The activated monomers are deoxyribonucleoside 3'-phosphoramidites. The meaning of activated is that the reactive group, hydroxyl group on the fifth carbon (5') is replaced by DMT, dimethoxytrityl. It keeps the deoxyribonucleoside 3'-phospohramidite molecule away from any further reaction on 5'. Thus, it was said to be a protecting group. The 3'-phosphoryl group is protected by a β-cyanoethyl (βCE) group. Since it is the 3'-phosphoryl group that is attached to the insoluble support, the chain is synthesized in 3' to 5'* direction. The synthesize of oligonucleotide consist three basic steps.


 * 3' to 5', meaning the 5'hydroxyl group perform a nucleophilic attack on the 3'-phosphoramidite of the incoming monomer. It is different from the regular 5' to 3' synthesize of natural DNA and RNA, where the 3' hydroxyl group attacks the inner most phosphorus atom of 5' triphosphate.

Procedure
Step one: The procedure starts with deoxygribonucleoside 3'-phosphoramitide (with DMT that was already added to protect the 5' and a βCE that was added to protect 3'-phosphoryl) react with a growing chain to form a phosphite triester group through coupling. This coupling reaction is driven by the nucleophilic hydroxy group on the 5' carbon of the nucleoside that is to be added attacking the electrophilic phosphorus atom on the existing chain with the -NR2, often diisopropylamine (a relative stable secondary amine), as a leaving group. The reaction is carry out in an anhydrous environment to avoid water hydrolyse the 3'-phosphoramidte causing the reaction go backward unexpectedly. And avoid water from reacting with the 3'-phosphoramidite of the incoming monomers. Once the water attack the 3'-phosphoramidite, it is no longer reactive in receiving the nucleophilic attack from the 5'OH of the growing chain.

Step two: Using Iodine, I2, the phosphite triester group oxidized to a phosphotriester group.

Step three: The DMT group on the 5' side of the activated monomer is removed by adding dichloroacetic acid (DCA, CHCl2COOH) while the rest of the molecule unchanged.

The oligonulceotide is now extended by one monomer unit and is ready to react with another incoming activated monomer. When the oligonucleotide of the desired length is synthesized, the final product can be obtained by adding ammonia (NH3) to remove all the protecting groups and remove the product from the insoluble support. Because no synthesis is perfect, not all of the growing oligonucleotide will react with the added monomers every time. Some oligonucleoides will be shorter than the other, for some nucleotides are missing. Thus, that considered to be impurities as they do not have the exact sequence carries the exact genes we wanted. The final product is the longest ones. The mixture of newly synthesized oligonucleotides can be separated by gel electrophoresis to get the desired product that was interested at the first place.



Removal of Protecting group
After all the synthesis steps, the mixture of product is then put into concentrated ammonium hydroxide, NH4OH, for an hour at room temperature. Next, the mixture with ammonium hydroxide is placed into an ice-bath and transferred to a vial that has a screw-cap afterwards. In order to remove the heterocyclic hase protecting groups, the solution in a vial is heated at 55 oC over night. Next day, the solution would be cooled in an ice-bath and evaporated off to dryness.

Analysis of Synthetic DNA
Four methods can be used to analyze synthesized DNA.

1. High-pressure (performance) liquid chromatography (HPLC).

High pressure liquid chromatography is a technique use to identify or separate compounds. It includes two basic forms, reverse-phase and ion-exchange. The separation and analysis of HPLC are based on the retention time between the stationary phase(chromatographic packing material) and mobile phase(the compounds needed to identify or separate) in the column.

Ion-exchange HPLC is based on the charges between stationary phase and mobile phase. The compound with same charges as stationary phase has a shorter retention time while it has a longer retention time with opposite charges. It is used for oligonucleotides of 10 to 20 bases of nucleotides in length. Two sets of ion-exchange HPLC is used for analyzing DNA. One condition is dNAPac column with 20mM Tris buffer/0.05% acetonitrile and 1M NaCl at a rate of 1.5 mL/min. Another condition is Resource-Q column with 10mM NaOH/80mM NaBr and 10mM NaOH/1.5M NaBr at a rate of 1.5mL/min

Reverse-phase HPLC is based on the affinity between stationary phase and mobile phase. The stationary phase of reverse-phase HPLC is non-polar, a non-polar compound has a longer retention time than a polar compound.

2. Gel electrophoresis

Gel electrophoresis is a technique use to separate DNA, RNA, or protein molecules. For oligonucleotides, 15~20% cross-linked gels are used with 7M urea. The gel electrophoresis takes over 3~6 hours at 50 to 60oC. To see oligonucleotides, a Molecular Dynamics Phosphorimager is used.

Gel electrophoresis can be used to analyze synthesized DNA. It also uses for the purification of synthesized DNA. A longer synthesized DNA is mainly used for cloning and hybridization. Non-full-length synthesized DNA formed during oilgonucleotide synthesis may interfere the applications, so purification is important. Gel electrophoresis is an efficient technique to separate full-length products from other shorter products.

3. MALDI-TOF analysis

MALDI-TOF is an ionization technique in mass spectrometry. It is used to identify compounds like oligonucleotides by their mass. Oligonucleotide is ionized and given a potential energy in the ion chamber of MALDI instrument. The oligonucleotide is then move to the detector of MALDI instrument by converting potential energy to kinetic energy. E=1/2mv2. Since E(energy) is given and v(velocity) is measured, m(mass) of the nucleotide can be determined and compared to value it should have.

4. Enzymatic degradation of oligomers

The products dissolved in the mixture solution of snake venom phosphodiesterase, alkaline phosphatase, and buffer solution. The reaction takes 4 hours at 37 oC. After that the mixture is heated to denature the enzymes. The mixture is then diluted with water and centrifuged. Finally, the mixture is analyzed by HPLC.

Advantages
The advantages of solid support synthesize of oligonucleoside is similar to the one of synthesis of polypeptides. Because the growing nucleotides is attached to a insoluble support, all the soluble impurities can be washed away without loosing any product.

Application of Synthesized Oligonucleotides
The ability to synthesize oligonucleotide with the exact sequences have important uses. A synthesized oligonuletide can be used in many DNA manipulation techniques.

1. DNA Sequence

A synthesized oligonucleotide with fluorescent tag can used to locate genes within a very long DNA molecule if the gene sequence in known. Because then we will able to synthesized a new oligonucleotides that is complementary to the gene sequence we want to locate. The labeled oligonucleotide use as probe is very important in exploring new genes. The probe can be used as primer to initiate replication of neighboring DNA by DNA polymerase that have not been map yet. With the fluorescent ability of the probe, we are able to tell where the new gene we are trying to map starts.

2. Polymerase Chain Reaction (PCR)

The polymerase chain reaction is the technique to amplify specific DNA. In order to amplify specific DNA sequences, pair of primers to hybridize with the target DNA sequences, four deoxyribonucleoside triphosphates, and a heat-stable DNA polymerase are needed. The specific primers for hybridizing the target DNA sequences can be made by Oligonucleotide Synthesis Technique.

3. Site-Specific Mutagenesis

The Site-Specific Mutagenesis or Oligonucleotide-Directed Mutagenesis can produce mutant proteins with single amino acid substitutions. The key of this technique is to prepare an oligonucleotide primer that is complementary to the DNA except the region that wants to change amino acid. The mismatch of primers of 1 of 15 bases is tolerable at an appropriate temperature. These specific oligonucleotide primers can be prepared by Oligonucleotide Synthesis Technique.

4. Large-Scale Synthesis

The Large-Scale Synthesis of oligonucleotide is almost same with the procedure above except a column used in the procedure. The Large-Scale Synthesis uses a packed column rather that a loosely packed cartridge.

5. Preparation of DNA microarrays on planar glass surfaces

DNA microarrays or gene chips can be used to analyze the pattern and level of pexpression of all genes in a particular cell or tissue. This technique can be done by placing oligonucleotides on a planar glass surfaces. The probes (oligonucleotides) are synthesized on the silicon chip. Thin silicon rubber capillaries are put on a lass slide and the probes are synthesized. Complementary DNA that is fluorescently labeled is hybridized to the gene chips that are made by oligonucleotides. This hybridized gene chips show the expression level for each gene.

6. Southern Blotting

Southern blotting is a technique to identify DNA molecules. In order to identify a DNA fragment with a specific sequence, a 32P-labeled single-stranded DNA probe, a specific DNA molecule made by oligonucleotide synthesis, is needed. The probe is hybridized with the complementary sequence of DNA sample. and the specific sequence can be visualized by autoradiography.