Characterizing nucleic acid

Details Characterizing nucleic acid Characterizing nucleic acid

2000-04-10

2001-11-06

Horlick, Kenneth R. (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100

Reexamination Certificate

active

06312904

ABSTRACT:

This invention relates to methods of nucleic acid sequencing. More specifically this application relates to high throughput methods of generating Sanger sequence termination ladders of multiple templates and methods of operating and analysing those ladders simultaneously.
Most nucleic acids of interest are large molecules which may be from a few kilobases to hundreds of megabases in length. Since current sequencing technologies only permit routine sequencing of fragments of about 500 to 600 bases in a single run, it is not possible to sequence such large molecules directly. A major cost in any large scale sequencing project is fragmenting large DNA molecules and isolating each sub-fragment to allow it to be amplified and sequenced. This sequence information must then be collated and analysed to determine the sequence of the source molecule. This is usually done by molecular cloning methods.
Cloning for sequencing is typically performed as follows. A large DNA molecule is fragmented, generally with a type II restriction endonuclease, to generate a ‘library’ of DNA fragments. These DNA fragments are then ligated into vectors that can be cultured in a biological host. Isolation of individual DNA molecules from the library is effected by limiting dilution of the culture of the host organism such that subsequent plating out of the medium onto agar culture dishes results in the growth of colonies of the host derived from a single organism bearing only one of the DNA fragments from the library.
Various strategies for high throughput sequencing have been developed which exploit the methods of molecular cloning. Typically a hierarchy of cloning is performed. Very large DNA molecules such as human chromosomes are typically cleaved using restriction endonucleases which cut rarely thus generating large fragments. These are cloned into vectors which can accommodate such large fragments which are then transfected into an appropriate host. Yeast Artificial Chromosomes (YACs) are often used for this purpose. These are transfected into
S. cerevisiae.
The vector sequences flanking a clone are known and these can be used to ‘end sequence’ a large clone, identifying short sequences that identify the termini of the clone. These can be used to generate oligonucleotide probes. These probes are used to screen libraries of clones. Overlapping clones may be identified by hybridising an oligonucleotide probe to blots of isolated colonies of the host organism. Pairs of probes from different clones which hybridise to a third clone, generally indicate that the third clone spans the gap between the clones providing the probes. A series of clones can thus be ordered identifying their positions in the genome. This identifies gaps and thus missing clones which can be subsequently isolated. Once an ordered library of clones has been generated, these clones can then be sequenced by sub-cloning the large clones into vectors which carry shorter fragments such as Bacterial Artificial Chromosomes or M13 phages. These sub-clones may be ordered by end-sequencing again or may be sequenced directly in their entirety.
A typical approach to the sequencing a library of clones derived from a large source molecule starts with a ‘shotgun sequencing’ phase followed by a directed ‘finishing’ phase. Shotgun sequencing uses random selection of the clones to be sequenced. The initial selections of a shotgun sequencing project generate a lot of unique clones but as the proportion of a library that has been sequenced increases, more and more clones are re-sequenced by random selection. This means that a considerable amount of redundant sequencing is done if one wishes to completely sequence a library by shotgun approaches. For this reason it is usual to perform an initial shotgun phase to sequence a pre-determined proportion of a library. Once this is done, contiguous sequences are identified from the sequences that have been determined. Once these ‘contigs’ have been identified, the sequences that flank the contigs can be used to identify and sequence clones that span the gaps between contigs. This finishing phase is expensive and relatively slow.
It would be desirable for the purpose of large scale sequencing projects to be able to automate the procedures required in the sequencing process. Unfortunately the processes currently used that are based on molecular cloning are amenable to partial automation using equipment that requires skilled operators. Furthermore the methods are slow. In order to reduce the costs of sequencing the genomes of organisms of commercial and scientific value, it would be beneficial to develop methods that fully automate the fragmentation and ordering of clones and to further automate the process of sub-cloning and sequencing of ordered clones.
Sequencing
Conventional DNA sequencing according to the Sanger methodology uses a DNA polymerase to add numerous dideoxy/deoxynucleotides to an oligonucleotide primer, annealed to a single stranded DNA template, in a template specific manner. Random termination of this process is achieved when terminating nucleotides, i.e. the dideoxynucleotides, are incorporated into the template complement. A ‘DNA ladder’ is produced when the randomly terminated strands are separated on a denaturing polyacrylamide gel. Sequence information is gathered, using polyacrylamide gel electrophoresis to separate the terminated fragments by length, followed by detecting the ‘DNA ladder’ either through incorporating a radioactive isotrope or fluorescent label into one of the terminating nucleotides or the primer used in the reaction. The main draw back with this technology is its dependence on conventional gel electrophoresis, to separate the DNA fragments in order to deduce sequence information, as this is a slow process taking up to nine hours to complete.
The separation of a Sanger Ladder by gel electrophoresis imposes limitations on the throughput and accuracy achievable for DNA sequencing. The polymerase reaction used to generate a Sanger ladder is simple and relatively fast and can readily be performed in parallel or even multiplexed in the same reaction. Various novel sequencing methods have been developed that are compatible with PCR and hence exploit automation using 96 well plate robotics and thermocyclers.
Gel electrophoresis works on the simple principle that a charged molecule placed between two electrodes will migrate towards the electrode with the opposite charge to its own. The larger the molecule is for a given charge the more slowly it will migrate towards the relevant electrode. Nucleic acids are poly-ions, carrying approximately one charge per nucleotide in the molecule. This means that nucleic acids of any size migrate at approximately the same rate ignoring frictional forces from the separation medium. The effect of frictional forces is related to the size of the molecule or in the case of nucleic acids, the length of the molecule. This means that nucleic acids are effectively separated by length. The role of the gel matrix is to provide frictional force to impede migration. The speed of separation is proportional to the size of the electric field between the two electrodes. This means that increasing the size of the electric field will reduce separation times, however the electrical resistance of the separation medium means that heat is generated as a result of the electric field and the heat increases with the electric field. The higher temperatures increase the kinetic energy imparted to the analyte leading to greater diffusion and band broadening. This reduces the resolution of the separation. Gels can be cooled but heat dissipation from a slab gel is limited by its surface/volume ratio which is essentially a function of the thickness of the gel. Thinner gels dissipate heat better but there is an additional effect of increased resistance. This means that in slab gel techniques using gels of 200 to 400 &mgr;m thickness heating becomes severe if the electric field strength is greater than 50 V/cm. Replacement of the slab gel electrophoretic steps is the most attr

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