PacBio P4-C2, P5-C3, etc. - What Do They Mean?

We had been pondering about those cryptic terms and found by asking some people around that the P stands for polymerase and C stands for chemistry. Therefore, P4-C2 means polymerase of fourth generation and chemistry of second generation.

Polymerase

That got us curious about what the actual DNA polymerase sequences are for 2nd, 3rd or 4th generation. Everyone else told us that those would be top secret information at PacBio and as unavailable as Coca-Cola formula. We do not like to take no for an answer, and had to contact our friendly NSA collaborator to get to the deepest secrets from the company. Here is what we found. This document explains various polymerase sequences -

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions and/or heterologous or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing.

Compositions that include modified recombinant DNA polymerases that include amino acid substitutions, insertions, deletions and/or heterologous or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing are a feature of the invention. Relative to a wild-type ?29 DNA polymerase, these modifications can include any one of, or any combination of: an L253 mutation and a mutation at one or more of T368, E375, A484, or K512; an E375 and K512 mutation, and a mutation at one or more of L253, T368 or A484; an I93 mutation; an S215 mutation; an E420 mutation; a P477 mutation; a D66R mutation; a K135R mutation; a K138R mutation; an L253T mutation; a Y369G mutation; a Y369L mutation; an L384M mutation; a K422A mutation; an I504R mutation; an E508K mutation; an E508R mutation; a D510K mutation; or at least one mutation or combination of mutations selected from those listed in Tables 6, 9 and 10. The modified polymerases can exhibit desirable features described in detail hereinbelow, e.g., reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fractions, increased closed complex stability, enhanced metal ion coordination and/or reduced exonuclease activity, etc.

There is lot more in the document to keep a biochemist busy for his entire lifetime.

Chemistry

How about chemistry? Our friendly NSA collaborator took us to the following top-secret documents -

Phospholink nucleotides for sequencing applications

The present invention provides labeled phospholink nucleotides that can be used in place of naturally occurring nucleotide triphosphates or other analogs in template directed nucleic acid synthesis reactions and other nucleic acid reactions and various analyses based thereon, including DNA sequencing, single base identification, hybridization assays, and others.

Recombinant Polymerases With Increased Phototolerance

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties include increased resistance to photodamage, and can also include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

[Method for sequencing using branching fraction of incorporatable nucleotides] (https://www.google.com/patents/US8530164?dq=pacific+biosciences&hl=en&sa=X&ei =v-9mUvCpCMr2iwKa9ID4Cg&ved=0CHkQ6AEwCQ)

Provided are methods for enhanced sequencing of nucleic acid templates. Also provided are reaction conditions that increase branching fractions during polymerization reactions. Also provided are compositions comprising modified recombinant polymerases that exhibit branching fractions that are higher than the branching fractions of the polymerases from which they were derived. Provided are compositions comprising modified recombinant polymerases that exhibit delayed translocation relative to the polymerases from which they were derived. Also provided are compositions comprising modified recombinant polymerases that exhibit increased nucleotide or nucleotide analog residence time at an active site of the polymerase. Provided are methods for generating polymerases with the aforementioned phenotypes and methods of using such polymerases to sequence a DNA template or make a DNA. Also provided are methods and nucleic acid sequencing systems for determining which labeled nucleotide is incorporated at a site during a template-dependent polymerization reaction.

Isolation of polymerase-nucleic acid complexes

Compositions, methods and systems are provided for the isolation of polymerase-nucleic acid complexes. Complexes can be separated from free enzyme by using hook molecules to target single stranded regions on the nucleic acid. Active complexes can be isolated from mixtures having both active and inactive complexes by initiating nucleic acid synthesis so as to open up a portion of a double stranded region rendering that region single stranded. Hook molecules are targeted to bind the sequences that are thus exposed. The hook molecules bound to active polymerase-nucleic acid complex are isolated, and the active polymerase-nucleic acid complexes released.

Generation of modified polymerases for improved accuracy in single molecule s equencing

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions and/or heterologous or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include reduced reaction rates at one or more steps of the polymerase kinetic cycle, increased closed polymerase/DNA complex stability, enhanced metal ion coordination, reduced exonuclease activity, decreased branching fractions, and the like. Polymerases that exhibit branching fractions that are less than the branching fractions of the polymerases from which they were derived, or branching fractions that are less than about 25% for a phosphate-labeled nucleotide analog, are also provided. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

More Details

Following papers and documents may be helpful to understand the technology better.

Real-time DNA sequencing from single polymerase molecules

http://nar.oxfordjournals.org/content/38/15/e159.long

http://nar.oxfordjournals.org/content/38/15/e159/F2.expansion.html

http://www.pacificbiosciences.com/img/press_release_assets/PacBio_Response_to_ Helicos_Complaint_090110_final.pdf


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Written by M. //