Targeted Variant Analysis via Sanger DNA Sequencing

WSLH Department: Cytogenetics
WSLH Test Code: 895M50
Availability: Monday-Friday 7:45 AM - 4:30 PM, Saturday 7:45 AM - 12:00 PM
Turn-around Time: Approximately 10-28 days, with an average of 10 days. (Reports are issued Monday-Friday 7:45 AM - 4:30 PM)
Recommended Uses: For the detection of specific targeted DNA variants.
Additional Tests Performed:
Patient Preparations:
Specimen Requirements: Blood: 4 ml (preferred) - 2 ml (minimum) whole blood collected in EDTA vacuum type tube.

Dried Blood Spot: At least one circle completely filled and dried on Whatman FTA™ cards.

Saliva: Isohelix™ Saliva Collection kit used according to manufacturer instructions.

Buccal: Stabilized buccal swabs collected with SafeCollect kit (Zymo) according to manufacturer instructions.

Fresh/Frozen tissue: Sample size at least 0.3 cm cubed

Skin biopsies: 1-2mm punch biopsy

DNA: 3-5 ug DNA in TE buffer at a concentration of 50-100 ng/uL. For best results, DNA should be treated with RNAse. DNA must be extracted in a CLIA-certified Laboratory OR a laboratory meeting equivalent requirements as determined by the CAP and/or the CMS.
Specimen Handling & Transport: Store and transport specimens at room temperature (may transport with coolant during hot, >85 degrees, weather). Specimens must be received by the laboratory within 24-48 hours of collection.
Collection Kit/Container:
Collection Instructions: Blood: Draw blood using aseptic techniques into a sterile EDTA vacuum type tube(s). Invert tube(s) to mix. If using larger tubes, draw to full volume to avoid over-treatment with anticoagulant.

Saliva/Buccal: Collect according to manufacturer instructions.

Fresh/frozen tissue: Tissue that is pale tan to brown should be avoided if possible as this indicates necrosis. A 0.3-0.5cm cubed section of each tissue type should be collected using aseptic procedures. Place the specimen in a sterile tissue vial containing transport media. If multiple tissues are sent, please place the placenta in one vial and the other tissue(s) in a separate vial to minimize contamination of the tissues.

For skin biopsies: Skin should be cleaned using alcohol, do not use iodine or betadine as these will compromise the cell growth in culture. Do a 1-2mm punch biopsy that goes full depth through the epidermis into the sub-cutaneous fat. Place the specimen in a sterile tissue vial containing transport media.

DNA extracted from peripheral blood, cord blood, buccal swab, saliva, fresh and frozen tissue (postnatal), dried blood spots. We DO NOT accept DNA from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue. Label with the patient name plus a second identifier. DNA concentration and volume must also be provided on the specimen label.
Unacceptable Conditions: Blood that is clotted or hemolyzed is not acceptable. Blood must not be frozen. Plasma and serum are not acceptable.
Requisition Form: Cytogenetics Lab Genetic Diagnosis Form #131
Required Information: Laboratory regulations require the following minimum information to be provided on the requisition form for a specimen to be accepted for testing: Patient name or unique identifier; date and time of collection, patient date of birth and sex, specimen type/site of collection, test request(s), reason for referral, clinician name and UPIN/NPI, and address for reporting results. Please be certain that name/identifier on the form matches that on the specimen label.
Results Include: Positive- apparently homozygous; Positive- heterozygous; Negative
Limitations: 1. Copy number variations are not detected with this assay;

2. This assay cannot detect mosaicism;

3. Nucleotide variation in primer binding sites can lead to allele dropout.
Additional Tests Recommended:
Additional Comments:
Methodology: An amplicon surrounding the targeted variant is PCR amplified. Sanger sequencing is performed on the PCR amplicons. Sequence data are generated by capillary electrophoresis analysis of the sequencing products.
Includes: Isolation and quantification of genomic DNA, followed by bi-directional Sanger sequencing of targeted variant region as described in the interpretation. Sequences are aligned to reference sequence (GRCh37/hg19). Variants are classified for pathogenicity using publicly available databases (NCBI, OMIM, UCSC Genome Browser, ClinVar, HGMD), internal database information and peer reviewed literature.
CPT Code: 81479
Price: For pricing information, please call 608-262-0402.
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