Method of Analysis
A swipe of mucous membrane cells is taken from the mouth lining; the swab is placed in a buffer solution so that the sample is not spoiled.
After the samples arrive and are registered with FamilyTreeDNA in Houston (USA), they are sent, along with any important additional information to the laboratory (Genomics Research Center) for processing.
To extract the DNA from the swipe sample, each sample is treated with an enzyme (proteinase K), causing the cells to shatter and releasing their DNA into the solution. The process is similar to that used in forensic samples taken from licked stamps or teeth. A piece of the stamp is added to a buffer-proteinase solution. All samples are incubated over night (heated to a temperature of 56 ºC) and agitated, so that the cells more easily break open.
The Barcode of a new carrying tray is scanned in for the DNA and placed in a pipetting robot. The individual samples of customers are likewise scanned in. The robot transfers 8 samples to one plate simultaneously for up to 96 samples for extraction. This plate already contains a solution in which there are minute magnetic particles. The DNA-magnet solution is thoroughly mixed, so that the DNA receives a negative charge and is then attracted to the positively charged magnetic particles.
The plate is set on a magnet stand and the buffer solution is carefully drawn off. The magnetic particles with the DNA are washed and then a “DNA fixing buffer” is pipetted into each of the 96 indentations. The plate is set on a heating block and heated to 65 ºC. This loosens the DNA from the magnetic particles and they go into the fixing buffer.
The carrying tray with 96 separate holding tubes (single tubes with blue caps) is opened. The buffer, which now contains the DNA, is transferred to the individual tubes for storage. The tubes are then closed.
The entire plate is scanned in. Each individual test tube has an individual barcode on the underside. Each sample number is clearly assigned to a barcode and stored in the REMP database. The plate with the DNA samples is stored at -20 ºC in the freezer system.
The samples to be analyzed are “requested” from the REMP database. Individual tubes from the stored plates are stamped in a “delivery plate“. The barcodes of the single tubes are scanned again to check for accuracy, and a prepared mixture of chemicals and dyes used in DNA-analysis (primer mix) is introduced to one of the 96 in the PCR plate by the robot pipettier. A small portion of the thawed out DNA-solution is pipetted into the PCR-mix and the plate is sealed with a foil. The plate is placed in a PCR-device (by the German company Eppendorf) and the program for DNA replication and “dying” is started. In a series of heating and cooling steps, the DNA is copied. After approximately three hours, the PCR is de-activated and the PCR-Products are ready for sequencing or for electrophoresis.
The PCR-products for analysis e.g. the mitochondrial DNA (mtDNA) have to be purified again before the electrophoresis, which is done by a robot from the company CyBIO of Germany. Finally, the PCR-products in up to 16 of the carrying trays with up to 96 samples each is transferred to the sequencer and placed in the device for electrophoresis.
In the device, each DNA-sample “walks through” a capillary filled with a gel (polymer) to which a positive electrical charge is applied (electrode). Small molecules can pass through the gel faster than larger ones. The DNA is sorted “by size” and recorded by a camera using the dye markings.
The sequence of red, blue, green, and black peaks yields the (person specific) mtDNA-sequence.
A second option is recording individual peaks (Short Tandem Repeats STR) e.g. in the analysis of a man’s y-chromosome haplotype.
The analysis data are evaluated with the support of specialized computer software and checked for accuracy again by a scientist. In a few uncertain cases or in the case of unusual results, the internationally constituted group of scientists who work at FamilyTreeDNA has to confer; the group is made up of biologists and biotechnologists some of whom have worked in the field of DNA-analysis and genetics for more than 15 years.