Gene Synthesis

We Specialize in Gene Synthesis for more than 15 Years......

Features

  • 100% Accuracy
  • Fastest turnaround time at NO EXTRA charge
  • Most competitive price
  • Gene optimization for FREE!
  • Most experienced
  • Strictly confidential

Gene synthesis is to create artificial double-strand DNA molecule with particular in vitro. It is a powerful tool in biotechnology and plays a pivotal role in the field of synthetic biology. The major concept of gene synthesis method is to assemble chemical synthesized oligonucleotides into long DNA molecule with custom sequences. To date, several methods have been developed. The most widely used method is the Polymerase Chain Assembly (PCA) method, which uses the same technology as polymerase chain reaction (PCR) to construct desired sequences from short single-stranded oligonucleotides.

Sangon Biotech offer fast and reliable gene synthesis service starts as low as $0.27/bp, (to be continued)

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How to Order

Download Gene Synthesis Order Form and email to genez@sangon.com and copy orders to tracey@sangon.com

1.Principle and Procedure of Gene Synthesis

Step 1. Design

For DNA sequence less than 1 kb, DNA sequence was broken into short overlapping single-strand oligonucleotide sequences by particular software or websites. Oligonucleotide sequences are calculated to keep a lowest mispriming. For constructs larger than 1 kb, <1 kb subfragments are synthesized first and could be joined later using either restriction digestion-ligation or other methods such as PCR.

If the synthesized gene is designed for protein expression, codon optimization could be performed to adapt the codon usage to certain species. Codon optimization can improve expression of heterogeneous genes. Extra restriction sites or undesired structures such as repeats can also be removed during sequence optimization.

Step 2. Oligo Synthesis

Oligonucleotides are synthesized based on solid-phase DNA synthesis technology. This step is easy and fast as commercial oligonucleotides can easily be obtained in all place.

Step 3. Assembly

In the initial assembly of the oligos, a set of overlapping oligos are mixed in a tube and the desired sequence is assembled in a thermocycler at the present of DNA polymerase and dNTPs. During the polymerase cycles, the overlapping oligos anneal to complementary fragments and then are extended by polymerase. Each cycle thus increases the length of various fragments randomly depending on which oligonucleotides find each other.

There should be numerous fragments exists including the desired full-length sequence.

Step 4. Amplification

After this initial construction phase, a pair of outermost primers used to perform a regular PCR reaction. Only the correctly assembled full-length sequence could be amplified, while other fragments are separated.

Step 5. Cloning and Screening

Final product with a desired length can then be purified by gel purification and be cloned into cloning vector (plasmid pUC57 as standard) by ligation or other methods. Each molecule in the amplification product are cloned into corresponding plasmid and be separated during bacteria transformation. So correct sequences and those with errors are separated in different clones. DNA sequencing is used to confirm the sequence and those bacteria harbor plasmid with correct DNA sequencing is chosen for plasmid preparation and later applications.

2.Gene Synthesis Services

Sangon Biotech is one of the leading gene synthesis supplier over the world. With an experience of ten years, Sangon Biotech has developed our own gene synthesis technology platform and established a comprehensive service process. Our generalized gene synthesis services including De novo gene synthesis, subcloning and mutagenesis. Special requests such as gene library construction can be discussed case by case.

2.1 Gene Optimization (if requested)

Codon optimization is adapt the codon usage of the desired gene to the codon preferences of the target expression organism. But it is not enough, our gene optimization provide better sequence features such as increased mRNA stablility, favorable GC content, reduced repeats and secondary structures, as well as optimized codon usage to obtain higher yields of protein.

2.2 De novo Gene Synthesis

It is the most commonly used gene synthesis service. As described in section 1, we can synthesis almost any kinds of DNA sequence from 50 bp-20 kb. If not specified, your gene fragments was cloned into pUC57 plasmid vector, at the Sma I restriction site or any desired restriction present in the vector. The final plasmid harboring the correct gene sequence will be sent to you in lyophilized powder with detailed data including sequencing results and quality control data. What you need to do is to reconstitute the plasmid and continue your subsequent experiments.

2.3 Subcloning

We can also get your gene ready to use in your downstream applications, such as protein expression directly. The synthesized fragments can be subcloned into various vector provided by you as well as pUC57. Subcloing alone is also acceptable, if you provide inserts and vectors, e.g complex constructions using multiple fragments, we can perform the subcloing step for you.

2.4 Mutagenesis

Site-Directed Mutagenesis is to make specific intentional changes to the DNA sequence of a know gene. The major goal of site-directed mutagenesis is to generate mutations that may produce rationally designed protein or other elements (e.g. promoter regions, terminator sequences) that has improved or special properties.

Our site-directed mutagenesis service introduces single or multiple mutations (substitutions, insertions, deletions, or truncations) into existing DNA sequences quickly and economically. We use a targeted PCR-based process to introduce genetic variation precisely where you need it, then clone the construct into the vector of your choice. All constructs are then 100% sequence-verified and documented to help assure that the DNA you receive is exactly what you’ve requested.

Frequently Asked Questions and Answers

Q: Why do I need gene synthesis service?

Sangon Biotech provide comprehensive gene synthesis services including de novo gene synthesis, subcloning, mutagenesis and more customed requests. You can get ready to use plasmid for your downstream applications. By choosing Sangon Biotech, you can save money (gene synthesis by Sangon is less expensive than traditoinal cloning or synthesis by yourself) and time (so you can spend more time on your experiment design and analysis, and more projects). What's more, gene synthesis make it possible to obtain DNA molecule with desired sequence that do not exist in nature.

Q: How do I know that I am getting the correct sequence?

Sangon has established tight quality control steps during the whole duration of gene synthesis. Final plasmid will be sequenced to ensure 100% accurate sequence with desired.

Q: How do you perform quality control?

Sequence is verified with Sanger DNA sequencing and the present and size of insert is confirmed by restriction digestion.

Q: What gene length can be synthesized?

Sangon Biotech is able to synthesize gene sequence from 50 bp to 20 kb, generally the primary synthesized fragments is range from 500 bp to 2 kb, but fragments could be further assembled to form large size.

Q: Can you synthesize sequences with high GC content or repeats?

Sequences with high GC content or repeats is a little complex to be handle, a higher cost and longer duration is required. We will carefully review every sequence, and provide a custom quote for every project, which includes turnaround time and price details.

Q: Do you have any criteria for complex genes such as high GC content sequences?

We do not have strict criteria for "complex genes", actually we do not have an efficient tool to check if a sequence is "easy" or "hard" to work with. According to our experience, a sequence will be considered as a "complex sequence" if it meets one or more of following criterias:

a. GC content >70% or < 40% in part of the sequence or the whole sequence;
b. Long sequence repeats exists in a high frequency. e.g. repeats (internal repeats, direct repeats or inverted repeat) have the length of more than 12 bp and the interval between repeats is less than 100 bp;
c. Homopolymers longer than 15 bp;
d. Sequences have a high similarity with some "toxic" gene. e.g. homogenous sequences from virus are reluctant to be cloned into common vectors.

Q: What is the standard vector does Sangon Biotech clone into?

Synthesized gene is cloned into the pUC57 vector if not specified.

Q: What restriction sites do you use when cloning synthetic genes into pUC57?

If not specified, synthesized fragments is cloned into pUC57 via Sma I or EcoR V, or any specific restriction sites within the MCS upon request.

Q: What antibiotic resistance gene is found in pUC57?

Ampicillin.

Q: My vector is a commonly used expression vector. Do you have it in stock?

Customers must supply vectors other than pUC57. If you need to clone your synthetic gene into a custom vector, please send us the vector DNA and pertinent information (i.e. vector size, cloning site, copy number, etc).

Q: How do you deliver synthetic genes?

Standard deliverables for synthesized gene is 2-5 µg of lyophilized plasmid containing your desired synthetic construct.

Q: What is the recommended DNA resuspension protocol?

Before opening the tube containing the DNA, please briefly centrifuge the tube. Lyophilized DNA could attach to the wall of the tube. Opening without centrifugation may cause DNA loss. The lyophilized plasmid is stable at -20°C for at least 1 year. Plasmid dissolved in TE is stable for at least 6 months at -20°C or 4°C. Plasmid dissolved in water is stable for at least 6 months at -20°C in the absence of nucleases. Be sure the water used is at neutral pH to avoid depurination.