Fly Paper:technical notes

This page is for technical details related to Fly Paper that would be distracting if included in the story itself.

branched DNA
'''Spoiler warning! Plot or ending details follow'''

The idea of branched DNA and a form of DNA with six different nucleotide subunits was introduced in the TV show The X-files. What is the point of branched DNA? In Fly Paper Dana Scully has been working on this mystery, but forces are at work who do not want the secret to be revealed. Dana suspects that the DNA with six different nucleotide subunits is alien DNA, but her investigations lead to the discovery that it is a tool for performing genetic engineering on humans. Once the structure of the branched DNA is finally revealed it is clear that it contains the four standard nucleotide subunits (A, T, G, C) and two additional subunits, pseudouridine (P) and methylguanosine (M). One other unusual feature becomes clear, the sugar along the backbone of this nucleic acid is deoxytagatose, not deoxyribose.

The problem of sequencing this type of branched DNA turned out to be fairly simple. The functional sequences of the branched DNA are always in two parts: sections of DNA with only A, T, G, and C and sections with only P and M subunits. The human Herpes virus-17 DNAse enzyme can selectively cut the branched DNA at the junctions between the ATGC and PM containing sequences, allowing conventional sequencing of the ATGC-containing sequences. The RNA polymerase of human coronavirus-57C4F can make an RNA copy of the PM-containing sequences, converting them into RNA sequences which can in turn be converted to standard DNA sequences by Taq DNA polymerase and other standard laboratory RNA-directed DNA polymerases.

The function of this branched DNA is inherent in its structure. When sequenced, the PM-containing DNA was found to always be of four simple types; poly-P, poly-M, MPPMMMPPMPMPMMPPPMMP and poly-PM. For gene transfer of a new gene into human chromosomes, the branched DNA construct used is: a poly-P branch followed by a copy of a target sequence (typically 50 nucleotides long) already in a human chromosome and a poly-M branch followed by the new DNA sequence to be inserted into the host chromosome. The end of the new inserted sequence is marked by a poly-PM branch.

In situations where an existing DNA sequence is being replaced by a new DNA sequence, the MPPMMMPPMPMPMMPPPMMP marker is included at either end of the target sequence in an unbranched configuration. The MPPMMMPPMPMPMMPPPMMP sequence acts as a signal for excision of the endogenous target gene sequence.

These gene splicing reactions are catalyzed by a set of proteins coded for by the gene transfer vector. All of these enzymes have been traced back to existing genes in various bacteria, but they all show signs of having been modified under laboratory conditions for enhanced molecular recognition of the various PM-containing marker sequences.

It should be noted that the branched DNAs that were used to perform genetic engineering on humans (such as Dana Scully) also contained a large amount of "structural" DNA containing a mixture of A, T, G, C, P and M subunits selected in molecular evolution experiments as structurally stable "spacer" DNA for the artificial mini chromosomes that were the actual gene transfer vectors. When sequenced by conventional means, the "structural" DNA regions of the vectors yield sequence data with gaps corresponding to the locations of the P and M subunits.

The structure of the branches depends on linkage of the branches through the number 4 carbon of the deoxytagatose subunits in the branched DNA molecules.

In Fly Paper, the reason for the existence of branched DNA in Dana Scully is deduced when analysis of biopsy tissue reveals that her brain cells now contain genes that were inserted by the branched chain DNA vector that was previously isolated from her.

The branched DNA described in the X-files episode One Breath as being inactive, represents the processed waste product of the branched chain DNA vector. The artificial mini chromosome of the vector is targeted to specific cell types by a virus. In the case of Scully, many copies of the virus were unable to cross the blood-brain barrier and slowly lost their structural integrity, leaving protein remnants and DNA fragments with PM branches in the circulating blood.