After the PCR reaction was complete, I watched as the sample was passed through a vacuum filter to remove any “trash,” remaining from the PCR reaction. Following the filtration, with a clean sample in hand, Jason explained that the analysis could proceed along two different paths, depending on the kind of question being asked.

If the analysis were looking for a small set of SNPs in a large number of people—for example, if one were screening for the poor metabolizer genotype of cytochrome p450 2D6—the next step would be to incorporate fluorescently labeled nucleotides in each sample. The labeled samples would next be applied to a DNA hybridization plate where they would form complexes with known sequences of complementary DNA. The plates would next be passed under a laser that would be refracted by the fluorescent dyes. A camera would record the variations in fluorescence and refraction and an algorithm would help determine what sequences were present. That, in short, is how a DNA lab handles high throughput analyses.

But the forensic sample we were analyzing was not a high throughput task. The attorney who ordered the test wanted a complete sequence of his client’s hypervariable region. To do that, my host used a technique called Sanger Sequencing, also known as the chain termination method or dideoxy sequencing. The other Doctor Eshleman–my son–explained that this technique is the most cost-effective and versatile technique for investigating small samples and exploring unknown variation in a particular short stretch of DNA.

Sanger sequencing is the invention of two-time Nobel Prize winner Frederick Sanger. Sanger’s first Nobel (1959) came for determining the amino acid sequence of insulin. The prize for the chain termination method came in 1980.

Sanger sequencing is an elegant technique that requires more description than I want to get into here. In very simple terms the technique involves splitting a single DNA strand into smaller pieces and then producing complementary strands that have a dideoxynucleotide inserted somewhere on the strand. The dideoxynucleotide stops any further DNA replication beyond its position on the strand.

Next, the mixture of labeled strands of various lengths is passed through a capillary electrophoresis machine, separating the fragments by mass. The sequencer is roughly the size of one of those little “cube” dormitory refrigerators and costs between $80,000 and $300,000 depending on the number of samples that it can analyze at a time. By comparing the length of a fragment and a fluorescent label attached to the dideoxynucleotide terminators on the various strands, the analyst (aided by a computer program) can determine an entire short sequence of bases. You can read more about this technique in Wikipedia.

I had to leave shortly after the Sanger sequencing began. I was very impressed with what I saw. A few days later I called back to clarify a few points and also to ask what my own DNA had shown.

“Your mitochondrial DNA is Haplogroup K,.” Jason explained. “Your mtDNA sequence for the first hypervariable region (HVI or HVSI [hypervariable segment] or HVRI) of the mtDNA D-loop differs from the “Cambridge Reference Sequence” (the CRS or “Anderson sequence”) at nucleotide position np 16224, np 16234, and np 16311.”

According to some authors, I share this sequence with ~8% of other maternal Ashkenazi Jews.This motif is rather rare among non-Jews in Europe. Other variants of haplogroup K are common (~10%) throughout Europe, but the particular variant likely speaks to a more recent bottleneck at or after the Jewish Diaspora out of the Middle East within the last few thousand years.

The Haplogroup K lineage is probably more than 16,000 years old. No surprises here. My grandmother got out of Belaruss to New York via Ellis Island in the early 1900s, just ahead of the latest pogrom. But that’s another story.

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