Oligonucleotide synthesis

Oligonucleotide synthesis is the non-biological, chemical synthesis of defined short sequences of nucleic acids. It is extremely useful in laboratory procedures covering a wide range of molecular biology applications. Automated synthesizers allow the synthesis of oligonucleotides up to 160 to 200 bases. Typically, synthesized oligonucleotides are single-stranded DNA molecules around 15-20 bases in length. They are most commonly used as primers for DNA sequencing and amplification, as probes for detecting complementary DNA or RNA via molecular hybridization, and for the targeted introduction of mutations and restriction sites, allowing for the synthesis of artificial genes.

Synthesis substrates

Oligonucleotides are chemically synthesized using phosphoramidites. A phosphoramidite is a normal nucleotide with protection groups added to its reactive amine, hydroxyl and phosphate groups. These protection groups prevent unwanted side reactions and force the formation of the desired product during synthesis. The 5' hydroxyl group is protected by DMT (dimethoxytrityl), the phosphate group by a diisopropylamino (iPr2N) group and a 2-cyanoethyl (OCH2CH2CN) group.

Sequential synthesis

In solid-phase synthesis, the 3' end of the oligonucleotide is bound to a solid support column on which all reactions take place. The 3' group of the first base is immobilized via a linker onto a solid support (polystyrene beads or similar). This allows for easy addition and removal of reactants. In each step, the solutions with the nucleotides for the next reaction are pumped through the column from an attached reagent delivery system and washed out before the next nucleotide is added. In modern synthesizers, reagent delivery and washing steps are controlled via computer based on the desired sequence. At the end of the synthesis program, the oligonucleotide is cleaved off the solid support and eluted from the column.


Microarrays

An interesting development of this technology has allowed gene chips to be made, where the probes are synthesized on the silicon chip, and not printed, allowing a higher resolution. This can be done via a mechanical mask where thin silicon rubber capillaries are put on a glass slide and the probes synthesized. More high-tech versions employ photolayable products and Photolithographic mask or micro mirrors. The 1cm2 surface of silicon is coated with a linker and a photo protecting group such as nitroveratryloxycarbonyl is used and the mask exposes to a lamp the spots that will receive the subsequent nucleotide: this step is repeated for all four bases, but only one correct one is added to the growing probes on each spot

DNA Evidence Gains Acceptance As a Key Tool in Robbery Cases

By Gautam Naik, as featured in the Wall Street Journal, June 18, 2008

When David and Dina Weller robbed yet another home in Denver in 2006, investigators traced the couple by analyzing saliva left on a cigarette butt at the crime scene. That DNA evidence connected the pair to a string of burglaries, and each got a 36-year sentence.

The payoff was immediate and huge: The annual burglary rate for the neighborhood they operated in fell 40%. For years, DNA forensic techniques have largely been used for serious crimes such as rape or murder. Now they are also being applied to lesser felonies -- such as car theft and burglary -- often with dramatic results.

That success also poses a dilemma for cash-strapped police agencies and local governments: Is an expansive new high-tech infrastructure worth the price for solving relatively minor crimes?

Some think law-enforcement agencies have more pressing needs for such sophisticated sleuthing. "Many jurisdictions have a backlog for solving rapes and other violent crimes," and typically those should be tackled first, says David Lazer, a professor at Harvard's John F. Kennedy School of Government and an expert in the use of DNA for crime-solving.

Still, that hasn't stopped a five-city pilot project that revealed promising results earlier this week, notably that DNA evidence can significantly increase the chance of netting a burglar. Though burglaries and car thefts have dropped in many U.S. cities because of better policing and other measures, a study suggests that DNA evidence can make a huge difference in helping capture and convict those guilty of property crimes.

"We found that twice as many suspects were identified, twice as many were arrested and more than twice as many were prosecuted," says David Hagy, director of the National Institute of Justice, part of the U.S. Department of Justice, which funded the scientifically-conducted and randomized study.

Research suggests that habitual burglars commit on average more than 240 burglaries each year, and often don't stop there. "People committing serious crimes usually start on smaller ones. So through this process you can get these people identified and in the system earlier," says Steve Allison of the National Law Enforcement and Corrections Technology Center at the University of Denver.

The university recently teamed up with five regional Colorado law-enforcement agencies and a prosecutor to create a DNA lab that deals exclusively with property crimes. The latest statistics from the Federal Bureau of Investigation show property crimes, including burglary, larceny, motor-vehicle theft and arson, were down 1.1% nationally in 2007.

Britain was one of the first countries to embrace the broader use of DNA evidence. Its Forensic Science Service has even tested mobile vans that can analyze samples from a crime scene in six hours, far faster than a traditional lab. That may help quickly identify and nab a burglar still lurking in the vicinity. In some areas, police have given DNA swab kits to victims of hate crimes, in case they are spat on or otherwise attacked again.

Inspired by Britain's example, the Justice Department funded its project in Orange County, Calif.; Topeka, Kan.; Phoenix; Los Angeles and Denver. For the study, biological evidence was collected at as many as 500 burglary and similar crime scenes in each location, between November 2005 and July 2007. Detectives investigated half of the cases -- the control group -- by traditional means. The other half was investigated using DNA leads as well.

Two key results: DNA is at least five times as likely to result in a suspect identification compared with fingerprints. Plus, suspects identified by DNA were found to have at least twice as many prior felony arrests and convictions as those identified in the control group.

There are considerable hurdles to expanding the use of DNA sampling in crime fighting. Civil-liberties groups fret that the rapid growth in DNA databases -- which studies say include innocent people along with offenders -- threatens to erode citizens' privacy. And DNA-based investigations are expensive and require trained police and high-tech equipment.

"What kind of bang for the buck are you getting?" says Tania Simoncelli, science advisor to the American Civil Liberties Union. "It's not a responsible use of our resources."

Another problem is more mundane: logistics. In a typical investigation, experts take a DNA sample obtained from a crime scene and try to identify the culprit by matching the sample with DNA profiles already stored in a database from previous crimes. But in forensic labs nationwide, a crush of DNA samples has caused huge backlogs. In many states, Harvard's Mr. Lazer estimates that it takes four to six months from when a rape or murder is committed to when investigators run a DNA sample through the database.

In Denver, property crimes had risen 5% annually for several years before the project started. Since then, police say they have used DNA to trap 95 prolific burglars, leading to a 13% annual decline in burglaries in each of the last two years. DNA evidence also more than quintupled the rate of case prosecution.

Were other factors responsible for the decline? As it turned out, one of Denver's six police districts didn't get involved in the project, "and it was the only area where crime levels rose," says Mitchell Morrissey, the city's district attorney and an advocate of DNA-based technology.

Antisense Technology

At Bio-Synthesis, we offer discovery scale synthesis to mid-scale multi gram quantities for clinical diagnostic applications, in addition to the synthesis of these modified oligos, we routinely assist customers in the design of the oligos that are particularly suited to their applications.

Antisense therapy
Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene "off". This is because mRNA has to be single stranded for it to be translated. The goal of antisense applications is to shut down activity of a defined gene by blocking transcription or translation within cells. Oligonucleotides for antisense assays must be nuclease resistant against cellular nucleases, must be able to cross cellular membranes and must inherit both high binding affinity and specificity for the target sequence. In many cases, they also must have the ability to induce RNase H cleavage.

Antisense DNA
Antisense molecules interact with complementary strands of nucleic acids, modifying expression of genes. Some regions within a double strand of DNA code for genes, which are usually instructions specifying the order of amino acids in a protein along with regulatory sequences, splicing sites, non coding introns and other complicating details. For a cell to use this information, one strand of the DNA serves as a template for the synthesis of a complementary strand of RNA. The stability of the RNA-DNA duplex in term of hybrization and half-life is crucial to successful gene inhibition. Vigorous research activity in the area of nucleic acid chemistry has been devoted in developing novel base analgos that are resistant to degradation and that possess strong hybridization properties. This includes the classical phosphorothioate linkages, propyne analogs and the latest lock nucleic acid (LNA) base analogs, and peptide nucleic acid (PNA).

Antisense mRNA
Antisense mRNA is an mRNA transcript that is complementary to endogenous mRNA. In other words, it is a non-coding strand complementary to the coding sequence of mRNA; this is similar to negative-sense viral RNA. Introducing a transgenic coding for antisense mRNA is a technique used to block expression of a gene of interest. Radioactively-labeled antisense mRNA can be used to show the level of transcription of genes in various cell types. Some alternative antisense structural types are being experimentally applied as antisense therapy, with at least one antisense therapy approved for use in humans.

Immunohistochemistry (IHC)

Immunohistochemistry

Immunohistochemistry is the localization of antigens in tissue sections by the use of labeled antibodies as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold. IHC combines anatomical, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label.IHC makes it possible to visualize the distribution and localization of specific cellular components within a cell or tissue. The term immunohistochemistry is often used interchangeably with immunocytochemistry and immunostaining.

Fixation
Tissue preparation is the cornerstone of immunohistochemistry. To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential. However, inappropriate or prolonged fixation may significantly diminish the antibody binding capability. There is no one universal fixative that is ideal for the demonstration of all antigens. However, in general, many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. The discovery and development of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories.

Sectioning
Paraffin wax has remained the most widely used embedding medium for diagnostic histopathology in routine histological laboratories. Accordingly, the largest proportion of material for immunohistochemistry is formalin-fixed, paraffin-embedded. Paraffin sections produce satisfactory results for the demonstration of majority of tissue antigens with the use of antigen retrieval techniques. Certain cell antigens do not survive routine fixation and paraffin embedding. So the use of frozen sections still remains essential for the demonstration of many antigens. However, the disadvantage of frozen sections includes poor morphology, poor resolution at higher magnifications, special storage needed, limited retrospective studies and cutting difficulty over paraffin sections.

DNA Hybridization

BIO-SYNTHESIS, INC., is a leading life science products company with over 20 years of experience in the design and synthesis of peptides, small molecules and reagents for small scale research and bulk pharmaceutical trials. Using state of the art technology in our well-equipped laboratories.

DNA Hybridization
DNA Hybridization generally refers to a molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences. It is usually used to determine the genetic distance between two species. When several species are compared that way, the similarity values allow the species to be arranged in a phylogenetic tree; it is therefore one possible approach to carrying out molecular systematics.

Nucleic Acid Hybridization
Nucleic Acid Hybridization is the process, discovered by Alexander Rich, of combining complementary, single-stranded nucleic acids into a single molecule. Nucleotides will bind to their complement under normal conditions, so two perfectly complementary strands will bind to each other readily. This is called annealing. However, due to the different molecular geometries of the nucleotides, a single inconsistency between the two strands will make binding between them more energetically unfavorable.

Peptide Antibodies

Bio-Synthesis has branched into several related areas such as DNA paternity testing, DNA HLA typing, PNA's, genomic sequencing, fluorescence based genotyping, custom organic synthesis and other molecular biology based applications. It has maintained its position as an aggressive, innovative company in a highly competitive marketplace without sacrificing quality. Today,newer technologies,such as synthesis of gene construction, PCR, mutagenesis, combinatorial libraries, dye/adduct labeling, DNA micro arrays, peptide-nucleic acid chimerics, etc, have challenged the molecular biology field.

Peptide Antigens
Well-designed anti-peptide antibodies provide the specificity and control for everyday and cutting-edge applications. Pragmatic peptide antigen design principles can be used to help ensure production of successful antibodies. Selective antibodies are powerful tools of experimental biology. Relatively straightforward immune blotting experiments can be used to identify a polypeptide antigen in a novel cell or tissue or to study cellular processing of a precursor protein that is required for biologic activity or targeting. Natural proteins are perhaps the ideal antigens, providing sequence-specific and surface structural epitopes. However, natural proteins are rarely available in completely pure form, and antibodies often develop against contaminating polypeptides. Antibodies against natural proteins, particularly monoclonal antibodies, sometimes have exquisite specificities, recognizing only subsets of the immunizing protein, when a more general reagent was desired.

Epitopes
An epitope, also know as antigenic determinant, is the part of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that recognizes the epitope is called a paratope. Although epitopes are usually thought to be derived from non-self proteins, sequences derived from the host that can be recognized are also classified as epitopes. Most epitopes recognized by antibodies or B cells can be thought of as three-dimensional surface features of an antigen molecule; these features fit precisely and thus bind to antibodies. Exceptions are linear epitopes, which are determined by the amino acid sequence (the primary structure) rather than by the 3D shape (tertiary structure) of a protein.

Custom Antibody Synthesis

For over 20 years, BioSynthesis has provided custom antibody production services worldwide, including researcher at university, biotechnology and pharmaceutical institutions. We offer comprehensive services for polyclonal antibody production with large selection fo host animals. Each of our facilities is operated under USDA license and hold an NIH Animal Welfare Assurance from the Office of Laboratory Animal Welfare.

Custom Antibody Synthesis
The specific suppressing activity of passively administered antibody on 7S antibody synthesis against sheep and chicken red blood cells has been investigated at the cellular level using the indirect hemolytic agar-plaque technique. 7S antibody production was found to be sensitive to antibody-induced suppression. No inhibitory effect of transferred antibody was seen until 48 to 72 hr after administration. This indicates that the action of antibody is not by direct suppression of synthesis of already committed cells but rather by removal from the system of the stimulus for maintenance of 7S synthesis.

Antigen Processing
When the macrophage eats bacteria, proteins (antigens) from the bacteria are broken down into short peptide chains and those peptides are then "displayed" on the macrophage surface attached to special molecules called MHC II (for Major Histocompatibility Complex Class II). Bacterial peptides are similarly processed and displayed on MHC II molecules on the surface of B lymphocytes.

Antibody Production
The stimulated B cell undergoes repeated cell divisions, enlargement and differentiation to form a clone of antibody secreting plasma cells. Hence. through specific antigen recognition of the invader, clonal expansion and B cell differentiation you acquire an effective number of plasma cells all secreting the same needed antibody. That antibody then binds to the bacteria making them easier to ingest by white cells. Antibody combined with a plasma component called "complement" may also kill the bacteria directly.

RNA Synthesis

With over 20 years experience in custom synthesis for the biomedical research communities, Biosyn has developed the expertise to deliver custom synthesized RNA with quality that meets all your RNAi, siRNA, sh RNA and other RNA projects.

RNA Synthesis
RNA synthesis is said to proceed in the 5′ to 3′ direction, reflecting the fact that the attachment of new nucleotides always occurs at the 3′ hydroxyl group of the growing RNA chain. RNA synthesis goes through phases that are typical of polymerization processes: initiation, elongation, and termination, yielding an RNA product of defined size and sequence.

Common RNA
Before the nucleotides are linked together, they exist separately as ribonucleoside tripolyphosphate (NTPs). As shown below, the NTP's contain one of the four common RNA bases, A, C, G, and U, linked to a five-carbon ribosome sugar, linked, in turn, to a chain of three phosphate groups. During RNA synthesis, a covalent, "phosphorescent" bond is formed between one of the three phosphate groups on one NTP and a hydroxyl group on another. The two other phosphate groups that were part of the original NTP are released.

Nucleotides
RNA, like DNA, is a polymer of nucleotides. Each nucleotide consists of a sugar that is attached to a phosphate group and any one of four bases. The RNA polymerase, as it builds the chain of nucleotides, processes only one of the two complementary strands of DNA. This DNA strand is referred to as the template strand. The least confusing name for the other DNA strand is "the non-template strand.