In principle, a DNA synthesizer can be used to synthesize the DNA sequence chemically and this can be cloned in the usual manner. But this is not so simple. A synthesizer will add bases sequentially one at a time to the growing oligonucleotide chain through a series of chemical reactions and washing steps. Synthesis of oligonucleotides 30-50 bases long is very reliable, longer sequences can be synthesized but the practical limit is not more than 100 bases. One way to solve this is to synthesize short fragments and join them chemically or enzymatically to create the longer fragment. However, the synthesizer makes single-stranded DNA, so the complementary strand has to be synthesized again to create a double-stranded DNA. It involves a lot of work but is achievable.
Early studies. The synthesis of nucleic acids in the laboratory started about thirty years ago. Early synthetic efforts used phosphodiester approach which enabled the synthesis of short oligonucleotides of 10-20 nucleotides. This approach was based on the selection of the proper condensing agents for phosphodiester bond formation and at the same time suitable protective groups were employed for the bases and the ribose moiety. These oligonucleotides were then assembled into longer DNA fragments with the help of kinase and DNA ligase. From the known primary structure of a ribonucleic acid, tyrosine tRNA, Dr H Khorana and his colleagues deduced the DNA sequence and synthesized successfully a DNA segment containing 200 bp coding for the structural gene for tyrosine tRNA. However, the low yields in the condensation step, the long reaction times, and especially the time-consuming purification of intermediates led to believe that chemical gene synthesis is unlikely to become a standard laboratory method.
Since then, the procedure for oligonucleotide synthesis has been improved by several workers and they provide different approaches for synthesis as well as protection of bases and sugar moieties. There are three distinct methods: (1) phosphodiester approach, (2) phosphotriester or phosphate triester approach and (3) phosphite triester or phosphoramidite approach.
Phosphodiester approach This method involves the formation of an ester linkage between an activated phosphate group of one nucleotide with the hydroxyl group of another nucleoside, thus forming the natural phosphodiester bridge between the 5?-OH of one nucleoside unit and the 3?-OH of the next.
Here, 3?-O-acetylnucleoside-5?-O-phosphate (a) is activated by N,N’-dicyclo- hexylcarbodiimide (DCC) or p-toluenesulphonylchloride(PTS/PTsCl) and subjected to react with a 5?-O-protected nucleoside (b) to give a protected dinucleoside monophosphate or phosphodiester (c). Activation of phosphate moiety is essential for easier formation of the phosphodiester linkage and this is mediated by DCC or PTsCl. Now, to increase the chain length, one has to remove the 3?-O-acetyl group by base catalysed hydrolysis. Further chain elongation is carried out by repeating the process. The major drawback of the phosphodiester method is the formation of pyrophosphate oligomers and oligonucleotides branched at the internucleosidic phosphate.
Phosphotriester approach In this method, oligonucleotide branch formation is avoided by protecting the phosphate group with an ethylcyano group. A nucleotide containing 5?-OH protected and phosphate protected by MMT and 2-cyanoethyl group respectively (compound “a”) is activated with 2,4,6-Triisopropylbenzenesulfonyl chloride (TPSCl) and subjected to reaction with a 3?-O-protected nucleoside (b). This generates a dinucleoside monophosphate or phosphotriester (c) in which phosphate group is protected by 2-cyanoethyl group. The basic difference between phosphodiester and phosphotriester method is that, in phosphodiester method, the phosphate group is protected by two phosphoester linkage but in phosphotriester method the phosphate group is protected by one extra phosphoester linkage with 2-cyanoethyl group. In phosphotriester method, the formation of oligonucleotide branch at the internucleosidic phosphate is avoided.
Phosphite triester or phosphoramidite approach The phosphite triester or phosphoramidite approach for oligonucleotide synthesis was based upon the use of phosphoramidite monomers and the use of tetrazole catalysis. In phosphite triester method, the starting compound is N-6-benzoyldeoxyadenosinephosphoramidite (if adenine is the first base) where the phosphorous atom is in the 3 oxidation state. So unlike the other methods, the formation of oligonucleotides branch is not possible in this process.
In this approach, the oligonucleotide is synthesized by a series of reactions described below.
Protection of base and sugar In this step, the free -NH2 group of the bases are protected by benzoylation or acylation depending upon the nature of bases. The 5?-hydroxyl group is also protected by dimethoxytrityl group (DMT), which protects only primary hydroxyl group but not secondary. The reactions are illustrated in CSG_Fig 3., the blocked bases are shown in the inset.
Formation of phosphite triester or phosphoramidite In this step phosphite triester is synthesized by a series of reactions. First, 2-cyanoethanol on reaction with phosphorus trichloride produces an intermediate compound which on further reaction with di-isopropylamine (two-equivalent) and 5?-OH protected nucleoside (one-equivalent) produces phosphite triester (CSG_Fig 4). This phosphoramidite will be repeatedly used during the oligonucleotide synthesis process described below.
The synthesis procedure The synthesis is carried out in several steps described below:
Step 1: The deblocking step
The first base, which is attached to the solid support, is at first inactive because all the active sites have been blocked or protected. The free -NH2 groups in the bases remains protected by benzoylation or acylation depending upon the bases and the -OH group is protected by dimethoxytrityl group (DMT). To add the next base, the DMT group protecting the 5?-hydroxyl group must be removed (deblocking). This step is also called detritylation. This is done by adding either dichloroacetic acid (DCA) or trichloroacetic acid (TCA) in dichloromethane (DCM), to the reaction column. The 5?-hydroxyl group is now the only reactive group on the base monomer. This ensures that the addition of the next base will only bind to that site. The reaction column is then washed to remove any extra acid and by-products.
Step 2: Base condensation
The step2 is basically a condensation step. Now prior to addition of the well protected nucleotide to the column, it is essential to activate the phosphate group, so that the nucleophilic attack on phosphorous atom takes place easily. This is best done by adding tetrazole to the nucleotide in dichloromethane medium. In presence of tetrazole, diisopropylamine group of the nucleotide becomes positively charged and hence its departure would be easier after nucleophilic attack of 5?-hydroxyl group of the previous nucleotide which is attached with resin column. After the reaction, the column was washed to remove extra tetrazole, unbound nucleotide and byproduct (diisopropylamine).
Step 3: Capping
In case of unreacted nucleoside attached with resin, the 5?-hydroxyl group is unprotected this may react later with the addition of different nucleotides. If left unprotected, it will lead to the formation of a mixture of oligonucleotides. The 5?-hydroxyl group is therefore blocked by adding acetic anhydride and N-methylimidazole (capping). After capping, the reaction column is thoroughly washed to remove extra acetic anhydride and N-methylimidazole.
Step 4: Oxidation
This step is basically an oxidation step. In this step, the phosphite linkage is oxidized to give more stable phosphate linkage. The oxidation is best done by adding a mixture of dilute aqueous iodine solution, pyridine (Py) and tetrahydorfuran (THF) to the reaction column.
The steps one through four, i.e., deblocking, base condensation, capping and oxidation, are repeated until all desired bases have been added to the column. This cycle is completed once for each additional base.
Step 5 Detachment of oligonucleotide from solid support
After all bases have been added the oligonucletide must be cleaved from the solid support and deprotected before it can be effectively used. For detachment of oligonucleotides form resin, the column is treated with 28% ammonium hydroxide solution (NH4OH), and at the same time the ethylcyano group on the phosphate group is removed.
Step 6: Purification and isolation of oligonucleotide
In this step, NH4OH is evaporated from the ammonium hydroxide solution of oligonucleotides to get crude product. The crude product is a mixture of oligonucleotide, cleaved protective groups and oligonucleotides with internal deletions. Now this crude product is subjected to boiling in a sealed tube with NH4OH at 55°C. The main purpose of this reaction is to remove the base protecting group. After evaporation of NH4OH, the crude product is subjected to desalting followed by Polyacrylamide Gel Electrophoresis, to purify the oligonucleotides. Desalting is used mainly to remove the ammonium ion. This is done by ethanol precipitation, size-exclusion chromatography, or reverse-phase chromatography.
Oligonucleotides are synthesized by the stepwise addition of nucleoside-3â€²-phosphoramidite monomers to solid-phase supports in an automated DNA synthesizer. In solid-phase synthesis, 3?-terminal hydroxy group of the first added nucleoside is attached to the solid surface by covalent interaction. The solid support is contained in columns whose dimensions depend on the scale of synthesis. The two most frequently used solid phase materials are Control Pore Glass (CPG) and macroporous polystyrene (MPPS).
CPG is commonly defined by its pore size, for example pore sizes of 500Å are used to allow the oligonucleotides preparation of about 50 -mer. To improve the performance of native CPG some modification is required. This is done by treating the material with (3-aminopropyl)triethoxysilane) to give Aminopropyl CPG. The amino group then serves as the anchoring point for the first added oligonucleoside.
MPPS is synthesized by polymerization of divinylbenzene, styrene, and 4-chloromethylstyrene in the presence of a porogeneous agent. It is a low-swellable, highly cross-linked polystyrene and suitable for oligonucleotide synthesis. The macroporous chloromethyl MPPS obtained is often converted to aminomethyl MPPS to improve the efficiency of the support.
Annealing of oligonucleotides For chemically synthesize a gene, the next step will be to assemble the oligonucleotides to form a complete gene. This is achieved by enzymatic methods which include polymerase cycling and ligase reactions. Some of the strategies are discussed below.
Assembling oligonucleotides by single-step PCR. For synthesis of a gene, the oligonucleotides (about 30-60 nt long) are synthesized chemically so that each oligonucleotide has a 6-9 nt overlap with its neighboring oligonucleotide. These are then assembled in a single-step PCR. In this method, oligonucleotides are first ligated and then the product, the entire gene, is PCR amplified using the outmost oligonucleotides as primers.
This method was first used to synthesize a 924-bp gene coding for an isozyme of horseradish peroxidase. Another method was developed by WPC Stemmer which did not use any ligase for joining the oligonucleotide products. It however, relied on Taq DNA polymerase (PCR cycling) for joining the individual oligonucleotides.
Assembling oligonucleotides by two-step PCR. The method involves two steps. (i) Synthesis of individual fragments of the DNA of interest: ten to twelve 60mer oligonucleotides with 20 bp overlap are mixed and a PCR reaction is carried out with high-fidelity DNA polymerase Pfu to produce DNA fragments that are 500 bp in length. (ii) Synthesis of the entire sequence of the DNA of interest: five to ten PCR products from the first step are combined and used as the template for a second PCR reaction using high-fidelity DNA polymerase pyrobest, with the two outermost oligonucleotides as primers.
Several modifications of the above procedure have been presented. One such method called PAS (PCR-based accurate synthesis) involves (i) synthesis of oligonucleotides to cover the entire DNA sequence (ii) PCR to synthesize DNA fragments (iii) second PCR for assembly of the products of the first PCR and (iv) cloning of the synthetic DNA and then verification by DNA sequencing.
Besides, other methods in use for gene synthesis are successive extension PCR, simplified gene synthesis (PCR based), synthons and ligation by selection, to name a few.
Review questions and problems What is the advantage of phosphatetriester method over phosphatediester method?
What is the advantage of phosphitetriester method over phosphatetriester and phosphatediester method?
What is the main advantage to use DMTCl for protecting the 5?-hydroxyl group?
How could you attach the first nucleoside to the solid support?
What is the utility of capping step in the oligonucleotides synthesis?
Why capping is done by aceticanhydride?
What is the function of iodine in the oxidation step of oligonucleotides synthesis?
How could you protect only the free -NH2 group of the bases of a nucleoside?
What is the reagent used for the removal of 2-cyanoethyl group from the synthesized oligonucleotides?
What is the byproduct produced from the base-condensation step of oligonucleotides synthesis in phosphite triester method?
How could you deprotect the bases of oligonucleotides?
What is the function of tetrazole in the base condensation step of oligonucleotide synthesis?
What is the basic principle for synthesizing a gene from the corresponding oligonucleotides by (a) PCR-based one-step DNA synthesis, (b) PCR-based two-step DNA synthesis?
Coal Workers’ Pneumoconiosis (CWP) Research Plan
Collection of Data
A.1. What were the objectives of the study? What was the association of interest?
The objectives of the study were to examine the association of the prevalence of coal workers’ pneumoconiosis (CWP) in the USA and different contributing factors such as level of dust exposure, mine size, low seam mining and other factors. Also the regional differences in CWP were compared. The association of interest is between CWP and various contributing factors and also between different MSHA district regions. It was a cross-sectional study.
A.2. What was the primary outcome (usually a disease, health condition, or other dependent variable) of interest? Briefly explain how the outcome was measured.
The primary outcomes were observed and predicted prevalence in CWP prevalence in miners who participated in this study. Attfield and Morring Exposure response model was utilized to measure the predicted prevalence in CWP and the Chi-square test was used to compare the predicted and observed prevalences in CWP in miners. Radiographs were used to determine the presence of lung parenchymal abnormalities that are consistent with pneumoconiosis obtained from CWHSP.
A.3. What was the primary exposure (actual exposure such as chemical, other risk factor, or other independent variable) of interest? Briefly explain how exposure was measured.
The primary exposures were level of dust exposure concentration, mine size, tenure, seam height. These data were collected from CWHSP, approved by the NIOSH Human Subjects Review Board. Coal mine dust concentration and seam height data were obtained from MSIS.
A.4. What type of study was conducted (study design)? This was a cross sectional study, which was done with a large number of participants from survey data.
A.5. Describe the process of subject selection. Define the source population for this study, if possible.
The study participants were 12,408 underground coal miners from the MSHA districts. Participation in this CWHSP study were voluntary and study participants were 16 years and older. Due to small number of participants and different type of coal type (anthracite rather than bituminous), MSHA district 1 participants were excluded. The source population was all the underground coal miners in MSHA districts in USA.
A.6. Selection bias: What are possible sources in this study? Examples: in a case-control study, how were the study subjects included? In a cohort study, is there loss to follow-up?
The study participants (underground coal miners) in this CWHSP study were stratified by MSHA
districts. Since stratified analyses are important in this study, the probability of being selected in a specific stratum might be different from another stratum and thus a selection bias might have happened.
A.7. Information bias: What are some of the sources of information (measurement) error, for either the exposure or outcome measurement? Are these differential with respect to the exposure or outcome of interest?
For exposure measurement, the investigators analyzed the self reported tenure in mining to derive the cumulative exposures. Also current exposures were examined for CWP which may not establish temporal relationship due to lack of allowance for lag time.
A.8. Confounding: Did the authors consider potential confounders in the design of the study?
The authors considered the following confounders: miner age and coal rank because the effect of respirable coal mine dust can be modified by the rank of the coal.
B. Analysis of Data
B.1. What methods were used to control confounding? Were these sufficient (as far as you can tell)?
As mentioned in the report the investigators incorporated all of the above mentioned covariates in their exposure-response statistical models to control for confounding. No detailed description is found about controlling the confounders.
B.2. What measure of association (e.g. odds ratio, risk ratio, rate difference, etc.) was reported in the study? Was this appropriate?
The authors reported the prevalence of the CWP and prevalence ratio between different MSHA district regions in this study. Since this is a survey based study, prevalence ratio is appropriate.
B.3. How was the uncertainty of the measure of association (effect of random error or statistical significance) reported in this study? Are the conclusions of the study consistent with the uncertainty of the measure of association?
The authors did not report 95% confidence intervals to report the uncertainty of the measure. However they reported the range of measured dust concentration level, worked hours per miners and tenure median. The prevalence ratio was statistically significant (p<0.05) in terms of measure of association between different MSHA regions.
C. Interpretation of Data
C.1. What was the major result of this study?
Major result of this study was that the authors found a significantly higher prevalence of CWP in central Appalachia (MSHA districts 4-7: mainly southern West Virginia, central and eastern Kentucky,
western Virginia and Tennessee) than the predicted levels (10.1% vs 4.2%). However, the prevalence was significantly lower than predicted levels in other regions (1.6% vs 3.6%).
C.2. How would the interpretation of this result be affected by bias (e.g., selection bias, information bias, or confounding)?
There are some factors that might have contributed to the findings of the study. For example, the workforce in central Appalachia was older than other regions. Also central Appalachia had lower proportions of mines with 200 or more workers. Moreover the average seam height was lower in central Appalachia than other regions. Stratification might have affected the result along with the self reporting of study participants.
C.4. To what larger population may the result of this study be generalized? Justify your restrictions (if any) to the generalization of these results
The result of the study may be generalized to all the coal workers in the USA. It might also be generalized to other parts of the world where the coal miners suffer from similar health effects.
C.5. Did the discussion section adequately address the limitations of the study? Was the final conclusion of the paper a balanced summary of the study findings?
The authors discussed limitations of the study adequately. For example, the use of personal protective equipment was a limitation in this study which might have some effect on the result. Also, silica was not considered in this analysis which is supposed to be an important factor. So, the final conclusion of the paper was a balanced summary of the study findings.
C.6. Given the results of this study and the initial literature review, what would you recommend as the next study or intervention to conduct addressing the exposure-disease relationship evaluated in this study?
A future study could be done considering other important factor like silica dust exposure. Other than survey data, an observational study can be done comparing different levels of exposure at different MSHA districts regions.