Non-Invasive Genotyping

Traditional genotyping requires the use of DNA isolated from harvesting fresh tissue samples from laboratory animals-a procedure known to cause pain and stress to the animal. For most research institutions, it is an ethical and legal obligation to reduce or eliminate pain and distress in research animals. First published in 1959, Russel and Burch’s The Principles of Humane Experimental Technique describes the 3Rs of a new applied science with the intent to improve the treatment of laboratory animals. These terms: replacement, reduction and refinement are the key tenets to guide minimizing animal pain and distress in biomedical research.

Refinement, as defined in the Principles is concerned not only with minimizing distress during experiments, but with maximizing comfort and wellbeing of the animals in husbandry. Eliminating the need for painful tissue excision for the purposes of genotyping is one way to meet this ethical obligation when working with research animals. To this end, we have expanded our genotyping platform to include the ability to screen DNAs isolated from client submitted buccal swabs, a non-invasive procedure that can allow for re-sampling of mice in the absence of painful tissue excision.

Benefits:

Ability to re-confirm genotypes of a mating without the need for anesthesia or analgesia.
Ability to test for recombination events, at multiple timepoints, when using ubiquitously expressed conditional systems, without having to excise multiple tissue samples.
Minimize pain and distress in laboratory animals and comply with institutional IACUC regulations.
Reduce time and costs associated with obtaining multiple tissue specimens.

Process Validation Data:

Publication:

Lui Dvm MF, Osborne Ms M, Dehm Ma T, Lee Ba M, Castaneda Dvm PhD Daclam JA. Comparing Genotyping Accuracy Using Buccal Swabs versus Tail Biopsies by PCR in B6;C3-Tg(Prnp-SNCA*A53T)83Vle and B6;C3-Tg(Prnp-SNCA*A53T)83Vle Sncatm1Mjff Mice. J Am Assoc Lab Anim Sci. 2024 Apr 22. doi: 10.30802/AALAS-JAALAS-23-000045. Epub ahead of print. PMID: 38649258.

Limitations for Transgene Zygosity:

Qualitative PCR assessment of transgenics (indicating the presence or absence of a transgene) can be performed with DNA isolated from buccal swabs. However, this platform is not yet optimized for quantitative PCR to determine zygosity (i.e., hemizygous, homozygous, or negative genotype) of transgenes.

All other targeted mutations, point mutations, and endonuclease mediated mutations can be screened using DNA isolated from buccal swabs.

Assay Validation:

For new assays requiring validation, please ship a confirmed positive control (tissue sample) first. DO NOT ship any buccal swab samples until the assay has been validated. We will notify you when the assay has been validated and swab samples can be shipped.

For assays that have already been validated, you may ship swabs to our laboratory and the results will be delivered within 2-3 business days.

Submission Requirements:

Use only DNA-free Puritan™ PurFlock Ultra 6″ Ultrafine Micro Flock Swab w/Polystyrene Handle, 100mm Breakpoint, for buccal mucosa swab samples taken from mouse cheeks.
Swabs should be shipped in covered Axygen^® Deep Well Plates and packaged in a way that preserves individual swab identity and prevents cross contamination.

Sampling Best Practices:

Refer to your institution’s IACUC protocols concerning animal handling and sample collection prior to proceeding.
Swabs containing blood may inhibit PCR results and are not acceptable for genotyping.
For the best DNA yield it is preferred that samples be shipped overnight

Sampling Procedure:

Open the outer packaging of the swab. Preparing swabs by placing them in the appropriate well in a deep well plate can avoid confusion between swabs. If tissue samples are being submitted along with swab samples, they MUST be submitted on separate plates.
Hold the mouse securely and insert the brush-like end of the swab into its mouth. Rub the swab on its inner cheek by rotating the swab three times around its axis, taking care to avoid any facial veins or the tongue. This step is critical to ensure sufficient material for downstream DNA extraction. Note that you are collecting cell material from mucosa and not just a saliva sample. Please DO NOT submit swabs contaminated with blood as these inhibit PCR and will not produce reliable results.
Remove the swab from the mouse’s mouth and place the brush-like end of the swab into deep well plate. Release the distal part by cutting with scissors.
Cover plate securely.
For new assays requiring validation, please upload your assay information into your online account and ship a tissue sample from a positive control a the sealed 1.5ml tube. We will notify you when the assay has been validated and swab samples can be shipped.

Procedure Guidance:

Play Video

Shipping:

Log into or create your account and submit sample information
For new assay validation ship a well-sealed tissue sample from a confirmed control at room temperature in the provided below. We will notify you when the assay has been validated and swab samples can be shipped.
Ship to:

GenoTyping Center of America
10 Water Street, Suite 215
Waterville, ME 04901

Supplies:

Puritan Medical Products, PurFlock Ultra 6″ Ultrafine Micro Flock Swab w/Polystyrene Handle, 100mm Breakpoint
- Supplier No: 3318-U (nonsterile), VWR Catalog No: 89194-888
- Supplier No: 25-3318-U (sterile), VWR Catalog No: 89194-898
Axygen® Deep Well and Assay Plates (96-well, 1.1 mL)
- Supplier No: P-DW-11-C (nonsterile), VWR Catalog No: 10011-940
- Supplier No: P-DW-11-C-S (sterile), VWR Catalog No: 47734-788

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