1. How should we approach the synthesis of highly complex genes?

2. In the E. coli expression system, which stop codon is preferred?

In E. coli, the frequency of use is: TAA > TGA; TAG is rarely used.

3. In mammalian expression systems, which stop codon is preferred?

In mammals, the frequency of use is: TGA > TAA > TAG.

4. What is codon preference?

Codon preference refers to the fact that different organisms use degenerate codons at different frequencies.

5. What is codon optimization?

Codon optimization is a technique that enhances protein expression levels in organisms by increasing the translation efficiency of the target gene. Typically, genes are redesigned using methods such as avoiding rare codons, utilizing preferred codons, simplifying the secondary structure of mRNA, optimizing repetitive sequences, eliminating restriction enzyme sites, and adjusting GC content to improve translation efficiency and, consequently, protein expression levels.

Generally, a CAI < 0.80 is considered to require codon optimization.

6. What is CAI?

In the late 1980s, scientists developed the Codon Adaptation Index (CAI) based on codon usage frequencies reported in the literature for highly expressed genes.

7. What are the selection criteria for restriction sites in COA’s restriction digestion validation?

Restriction enzyme verification is a method used to confirm the correctness of a plasmid. When performing this verification, the first restriction site is prioritized; dual restriction digestion is preferred, provided the two bands are of different sizes. If a fragment is too small to produce a distinct electrophoresis pattern, or if the separated bands are too close together to be easily distinguished, other sites that best reflect the plasmid’s characteristics are selected.

8. What is a frameshift mutation? How can frameshift mutations be avoided when designing gene synthesis or subcloning protocols?

Since three nucleotides code for one amino acid, a frameshift mutation occurs when the number of nucleotides in a gene is increased or decreased by a number that is not a multiple of three. This causes a frameshift in the DNA sequence following that site, resulting in the expression of a protein other than the intended one. When designing protocols, we should note the following:

a. Selection of restriction sites, such as NcoI (CCATGG). Because this site contains a start codon, a frameshift mutation can easily occur. Therefore, caution should be exercised when using such restriction sites, and frameshift-protective bases may be added;

b. Utilize existing sequences on the vector as much as possible.

9. Which types of vectors are more difficult to subclone?

It is difficult to subclone large vectors, low-copy-number vectors, vectors with unclear backgrounds, suicide vectors, and vectors with specific resistance traits. Examples include pMG36e (erythromycin-resistant) and pCYT (chloramphenicol-resistant).

10. What primarily determines the success rate of gene synthesis? What factors should we consider?

The probability of successful synthesis is primarily determined by DNA structure (factors such as repetitive sequences and GC content) and host cell stability (factors such as toxicity). To increase the success rate of recombinant gene synthesis, the following factors must be considered.

Key factors include:

a. Overall GC content that is too low (below 20%) or too high (above 80%);

b. Variations in GC content within the DNA sequence;

c. Homopolymers (e.g., polyA);

d. Repeat sequence length ≥ 20 bp;

e. The number and length of short repeat sequences;

f. Presence or absence of palindromic sequences;

11. What is methylation?

Methylation is a form of chemical modification of DNA and an epigenetic modification that alters genetic expression without changing the DNA sequence.

12. What is included in the gene synthesis delivery?

-- Gene COA document

-- 5 µg of lyophilized plasmid DNA

-- Plasmid structure diagram

-- Sequencing map