Multiplexed Gene Fragments

There are no complexity requirements for MGF. However, some features may cause suboptimal results:

1. Features like direct repeats and conserved sequences shared across all variants may result in some recombination during amplification.
2. Long homopolymers (>20bp) may result in some drop out during amplification.
3. Strong hairpins may result in poor amplification, which may lead to underrepresentation or dropout of those variants.

No, all fragments in the pool must have the same flank sequences. Multiple sets of flanks can be accomplished by ordering multiple pools. Alternatively, you can design a single pool with internal flanks that are different and external flanks that are universal for Twist to use for amplification. You may then amplify out the subpools; although this does come with inherent risk of skewing uniformity and/or running out of template material.

No, we can only synthesize sequences containing the four standard bases A, C, G and T.  The use of degenerate bases (R, Y, S, W, K, M, B, D, H, V, N) are not allowed. However, if you would like to have all four bases (or a subset of bases) at a given position in a fragment, you can design separate fragments, each with a different base at the site of interest.

Yes, we can add standard Twist flanks to your fragments that can be used with restriction cloning.

Step 1: Download the MGF Submission Form and fill out all fields in the form for your MGF design.  

Step 2: Upload the completed submission form here.  

If you have any questions or issues, please contact us via email (customersupport@twistbioscience.com) or chat.

Please consider the standard rules for primer design:

1. Length of 20-25 bp
2. 35-70% G/C content
3. Melting temperature (Tm) of 55-60°C
4. Primer pairs should have a Tm within 5°C of each other
5. Primer pairs should not have complementary regions
6. Avoid internal primer binding sites in the variable region

1. Fragment length can be between 301-500 nt (including constant flanks).
2. The total length of the fragment cannot exceed 500 nucleotides including constant flanks which will be used for PCR amplification. Flanking sequences must be at least 20-25 bps in length, which will allow for up to 460 bps of variable coding region. 
3. The variation of fragment lengths from shortest to longest should not exceed 80 bp (e.g. if the longest fragment is 400 bp, the shortest fragment should be > 320 bp).

1. Capillary electrophoresis is performed on the final pool to determine purity of the sample. At least 90% of the molecules in the pool must fall within the expected length.
2. Fluorescence-based quantification is performed on the final sample to ensure that there is at least 200ng of material.
3. Twist does not perform any sequencing on the pool, and does not guarantee quality in terms of representation or error rate. However, we have seen during development that most pools have a 90/10 percentile of < 3 and an average error rate of ~1:2,000.

The turnaround time for Multiplexed Gene Fragments is 8-12 business days.

Pricing depends on the complexity tier (Pool Size) and the fragment length tier (301-350nt, 351-400nt, 401-450nt and 451-500nt).

Multiplexed Gene Fragments are provided as amplified double-stranded DNA, as a lyophilized product pooled in a single tube

1. Unmodified multiplexed gene fragments are stable. For long-term storage (up to one year), freeze DNA at -20° C. For even longer storage, freeze DNA at -80° C.
2. If the fragments are stored in solution, avoid repeated freeze-thaw cycles as this process can lead to physical degradation of the fragments.
3. If numerous experiments are planned, aliquot the sample and store at -20°C.
4. Careful handling is recommended to avoid possible contamination with nucleases or bacteria.

Resuspension Guidelines:

• For resuspension, briefly centrifuge the tube before opening and resuspend in nuclease free Tris-EDTA (TE) buffer, pH 8.0 or 10 mM Tris-HCl, pH 8.0 to the desired concentration.
• A concentration of at least 10 ng/μL is recommended for the stock dilution, but the optimal concentration will need to be determined based on your desired application.
• Prepare aliquots of the stock dilution and separate working aliquots to limit chances of contamination and to reduce the number of freeze/thaw cycles. Use working aliquots as soon as possible after preparation and minimize exposure to high temperatures.

Resuspended DNA

• Twist recommends storing Resuspended DNA for the following durations based on storage condition.
  • Room Temperature for 3 months.
  • 4° C for 12 months.
  • -20° C for 24 months.
  • -80° C for long term storage.

Twist Bioscience ships Multiplexed Gene Fragments dried down in tubes at ambient temperature. Double-stranded DNA is stable under most standard laboratory storage conditions. However, it is important to consider the following best practices to maintain the high quality of the DNA synthesized by Twist Bioscience.

• Twist recommends storing Dried DNA for the following durations based on storage condition.
  • Room Temperature for 3 months.
  • 4° C for 12 months.
  • -20° C for 24 months.
  • -80° C for long term storage.

Twist uses phosphoramidite chemistry for synthesis. Compared to conventional column-based synthesizers, our synthesis platform miniaturizes the synthesis process, which reduces reaction volumes and increases throughput. This process has been optimized over the years and now allows Twist to print pools that are up to 500 nucleotides in length. After synthesis, we have an optimized amplification workflow to generate the multiplexed gene fragments in a dsDNA format.

Twist’s Multiplexed Gene Fragments (MGF) offer a unique pooled format for up to hundreds of thousands of double-stranded DNA gene fragments, between 301 and 500 base pairs in length. This pooled format of fragments enables high-throughput screening applications such as prime editing, ultra-complex CRISPR-based functional screening, peptide and protein engineering, Ab discovery, and massively parallel reporter assays (MPRA), where cost per fragment can be a barrier to the desired number of variants.

Twist uses an optimized amplification workflow to generate the multiplexed gene fragments in a dsDNA format which mitigates the risk of poor amplification results as PCR amplification of the synthesized material can be very challenging.