Reminder on RFLP and SSR analysis (Background)

The boys in Implementation and Planning keep whining about not understanding the jargon we keep using, so I had an intern write up a short summary of the more mundane technology we're talking about. If you have questions or complaints, just e-mail me and I'll be sure the little twerp gets an F. If it's really bad, then I'll just have Bill register him as a sex offender after we send him back to college.

Humans are a diploid mammalian eukaryote, with 22 pairs of autosomes and 1 pair of sex chromosomes, with gender determined by Y-mediated “XY” determination. At present estimate, humans have approximately 20,000-25,000 genes, with only about 1.5% of the total length serving as protein-coding exons, with the rest being occupied by transposons, pseudo-genes, regulatory elements, tandem and interspersed repeats, as well as other, undiscovered genetic elements.

All genetic analysis relies on a technique called Polymerase Chain Reaction (PCR), which is used to amplify (exponentially copy) small samples of DNA. The section of DNA amplified can be targeted, since the DNA needs an RNA primer to begin copying. If the sequence of nucleotides flanking the gene or segment of interest is known, then RNA templates can be constructed that anneal to the flanking sequence.

When these primers, the DNA of interest, and taq DNA polymerase (the protein responsible for copying DNA. Taq DNA polymerase is a polymerase extracted from a thermophillic Archaebacteria found in the geysers of Yellowstone, and thus are not damaged by high temperatures) are mixed together, melting the DNA allows the RNA templates to bind to the flanking sequences, which allows the taq DNA polymerase to begin copying both strands of DNA. This produces two strands of DNA, both of which have one molecule of original DNA, and one molecule of newly created DNA. This process is repeated several dozen times, at which point, you have (2n - 2) molecules of new DNA, and 2 molecules of original DNA. This process can be altered in many ways allowing for sequencing of the DNA, copying of multiple sites, as well as finding out the rate at which the DNA reanneals or melts, to find the repetitiveness of the DNA, or the ratio of A+T:G+C composition, respectively.

Reminder on RFLP and SSR analysis (RFLP analysis)

RFLP analysis is the older, and less efficient version of genetic fingerprinting. It uses bacterial enzymes called “restriction enzymes” to cleave your DNA at specific sites called “restriction sites”. Restriction enzymes are hypothesized to be a bacterial counter to viral invasion by cleaving the phosphate backbone of DNA at specific palindromic sequences ranging from 4-12 base pairs (bp) in length. Since human DNA has many of these sites (simply due to statistical chance), treating a sample of human DNA with 1-2 restriction enzymes will create a distinct pattern of bands when the cleaved DNA is separated via gel electrophoresis.

However, since all human DNA is slightly different, and one mutation in the composition of a restriction site can render it into “normal” DNA (and visa versa) very few people have the same pattern of restriction fragments, and application of several different restriction enzymes and use of different segments of DNA will always allow for distinction between two people (unless they’re identical twins) in case the initial results are in doubt. However, this takes time, and some regions mutate quicker then others, so results can take longer to develop and may be more subject to doubt. Thus, another form of genetic fingerprinting has come into favor, which exploits a highly variable segment of DNA.

Reminder on RFLP and SSR analysis (SRR analysis)

SSR analysis exploits the presence of the highly variable tandem repeat in human DNA. This highly variable region contains short repeating sequences of nucleotides, the repeats usually only 2-5 bp in length. The typical SSR has a repeat length of 2-9 nucleotides. A polymorphism in the number of repeats when the repeating unit is longer (in the range of 10-60 nucleotides) is called a variable number of tandem repeats (VNTR). A genetic polymorphism resulting from differences in copy number of a particular SSR is called a simple sequence repeat polymorphism (SSRP, pronounced “surp”), although the term simple tandem repeat polymorphism (STRP, pronounced “strip”) is also in widespread use (Hartl & Jones, 2004).

In most cases, these sequences exist in non-coding regions, while in several cases, their presence in coding regions has been implicated in the manifestation of a genetic diseases, most notably, Huntington’s disease. However, for purposes of genetic fingerprinting, these sequences are highly valuable. Since these sequences tend to shrink and expand between generations, comparison of only a few of these regions between any group of individuals will usually allow researchers to distinguish the individuals using their DNA. Since, on average, the human genome has one SSR every 2kb of human DNA, or about 1.5 million SSRs altogether, it is easy to find sequences to compare. This is done by applying RNA templates of sequences flanking the SSR, and then amplifying the SSR via PCR to create enough DNA for visualization. Since with every repeat, the length of the resulting fragments increases, even highly similar species of fragments (with differences of only 1-2 repeats) can be easily separated using electrophoresis.

Of course, these techniques are mostly good for placing someone at the scene of an event. However, the Genomic Revolution – beginning with the sequencing of the Human Genome, and J. Craig Venter’s application of shotgun cloning technique to whole-genome sequencing – has much more to offer.

Stage 1 Declassified

Still no promotion; I’ve made sure I won’t be passed up again, though. Also, do we agree that this thing is useless unless we use it? I mean, I understand these classifications exist for a reason, but it sure would be nice to know what’s going on in the other sectors. I hear they finally fingerprinted you boys in the lab, Lamarck. Still hate those needles?

Well it’s just my luck that they lowered the classification level of the first stage in the great “Atypical Subversive Strategy” on the same day I didn’t get my promotion. You might have access to it too now, but just in case, I’ll post it here. (My commentary will be in italics.)

STAGE 1: Introduction via top .1% (5-7 years)

1.0 - Introduction

The ISCP, once it has established itself in the private sector, will begin a revolutionary new service targeted to the highest tax bracket. This service will present genetic fingerprinting and inclusion in the National Genetic Database as a means of assuring the health and continued well-being of one’s self and family. It will present fingerprinting as a painless and risk-free procedure. Inclusion will originally deliver only modest benefits such as:

• A higher line of credit.
• Lower Interest rates.
• Expedited airline security procedure.

1.1 - GID

These will be justified through the creation of the Genetic Identity (GID). The creation of the GID will be supplemented by several events intended to devalue a citizen’s current identity:

• Several high profile cases of identity theft will hit newsstands. [This is being handled by the CIA, I believe]
• Top CEOs will have millions stolen from their bank accounts; the money will not be recovered.
• A terrorist will hijack an airplane by successfully imitating a security officer.
• Authorities will find out, days later, that they have executed the wrong person.
• Several hundred low profile cases around the country, intended to make the message “hit home.”

It will be explained that a GID completely removes the risk of identity theft. Several months after these incidents, national security will be saved thanks to a government official having been fingerprinted earlier that year.

1.2 -

We understand that the general public will, nevertheless, continue to perceive the service as they would a yacht or a passenger trip into outer space – an unnecessary, ostentatious luxury. That is, until certain discoveries are made in federally sponsored laboratories:

• At this time we will unveil our cure for Huntington’s as well as any additional cures we have discovered.

In addition, several independent companies ill begin offering services such as:

• COR (Complete Organ Replacement)
• PBGI (Pre-Birth Genetic Information) [supposedly they can do this currently, but I’m sure there will be some technical or moral setback within the next few months]

Fingerprinting will be a prerequisite for all of the above. After several lives are saved due to these procedures, we will initiate stage two.