Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-04-04
2004-12-21
Meyers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C435S287200, C435S287300, C435S286500, C435S288700
Reexamination Certificate
active
06833242
ABSTRACT:
1. FIELD OF THE INVENTION
This invention relates in general to a method for molecular fingerprinting. The method can be used for forensic identification (e.g. DNA fingerprinting, especially by VNTR), bacterial typing, and human/animal pathogen diagnosis. More particularly, molecules such as polynucleotides (e.g. DNA) can be assessed or sorted by size in a microfabricated device that analyzes the polynucleotides according to restriction fragment length polymorphism. In a microfabricated device according to the invention, DNA fragments or other molecules can be rapidly and accurately typed using relatively small samples, by measuring for example the signal of an optically-detectable (e.g., fluorescent) reporter associated with the polynucleotide fragments.
More generally, the invention relates to a method of analyzing or sorting molecules such as polynucleotides (e.g., DNA) by size or some other characteristic. In particular, the invention relates to a method of analyzing and/or sorting individual polynucleotide molecules in a microfabricated device by measuring the signal of an optically-detectable (e.g., fluorescent, ultraviolet, radioactive or color change) reporter associated with the molecules. These methods and devices can also be adapted to analyze or sort cells or particles.
The devices and methods of the invention are advantageous, particularly in comparison with conventional gel electrophoresis techniques. For example, the invention provides less costly and more rapid equipment, can use smaller molecular samples, is less labor-intensive and is more readily automated. The invention is also advantageously flexible. Additional functions can be incorporated into the design as desired, such as in-line digestion, separation, etc.
2. BACKGROUND OF THE INVENTION
When DNA is broken into fragments using restriction enzymes, each of which cuts the DNA in a known way, the resulting DNA fragments or polypeptides of different sizes produce a unique pattern or profile which can be used to uniquely identify the source of the DNA molecules. In the invention, a reporter or other measurable signal varies as a function of molecule size, and in this way profiles based on size can be efficiently generated and compared, particularly on a small scale and in an automated or semi-automated fashion.
Methods enabling the matching of unidentified tissue samples to specific individuals have wide application in many fields. For DNA fingerprinting, commonly used methods include RFLP analysis (53, 54), variable nucleotide tandem repeats (55), and microsatellites (56). With the possible exception of monozygotic twins, each individual in the human population has a unique genetic composition which can be used to specifically identify each individual. This phenomenon has allowed law enforcement officials to use DNA sequence variation to determine, for example, whether a forensic sample was derived from any given individual. The fields of forensic and medical serology, paternity testing, and tissue and sample origin have seen increasing use of such techniques, including the forensic and diagnostic use of DNA sequence variation, e.g., statistical evaluations based on satellite sequences and variable number of tandem repeats (VNTRS) or amplified fragment length polymorphisms (AMP-FLPS). These methods are being used in crime laboratories, courts, hospitals and research and testing labs. Inclusion probabilities stated by the laboratories performing the analyses in such cases often exceed 1:1,000,000. That is, only one individual in one million is predicted, on a statistical basis, to have a given DNA “fingerprint” obtained by analyzing a pattern of DNA fragments generated according to these techniques.
The first implementation of DNA typing in forensics was Jeffreys' use of a multilocus DNA probe “fingerprint” that identified a suspect in a murder case in England. (55) In the United States, DNA profiling has been established using a battery of unlinked highly polymorphic single locus VNTR probes. (57) The use of these batteries of probes permits the development of a composite DNA profile for an individual. These profiles can be compared to databases, for example using the principles of Hardy-Weinberg to determine the probability of a match between a suspect and an unknown forensic sample.
Although these methods have markedly improved the power of the forensic and medical scientists to distinguish between individuals, they suffer from a number of shortcomings including a lack of sensitivity, the absence of internal controls, expense, time intensity, relatively large sample size, an inability to perform precise allele (gene pair) identification, and problems with identifying degraded DNA samples.
For example, the most frequently used method for forensic identification is the “Southern” hybridization technique, which has been widely used in forensic identification and medical diagnosis. Also called a “Southern blot,” this technique treats an extracted molecule (a DNA sample) with a restriction endonuclease, an enzyme that cuts a polynucleotide chain wherever a specific and relatively short sequence of nucleic acids in the chain occurs. Examples of well known restriction enzymes used in this way are the endocucleases HaeIII, EcoRI, HpaI and HindIII. In DNA fingerprinting, restriction sites are typically used to isolate VNTRs (variable number of tandem repeats), which are regions in which a short sequence of DNA has been repeated a number of times. The number of repeating units within these regions vary between individuals, and when cut with a restriction endonuclease result in multiple fragments of different size called] RFLPs (restriction fragment length polymorphisms). These fragments can be used as a “fingerprint” because they vary in number and size from one individual to another.
The resulting nucleotide fragments (i.e. the RFLPs) are separated by size via gel electrophoresis, in which different sized charged molecules are separated by their different rates of movement through a stationary gel under the influence of an electric current. Following electrophoresis, the separated nucleotides are denatured and transferred to the surface of a nylon membrane by blotting; the so-called “Southern Blot”. The Southern Blot is then incubated in a solution containing a radioactive single locus probe under conditions of temperature and salt concentration that favor hybridization. (A single locus probe is also called a “primer.”) The locations of radioactive probe hybridization on the Southern Blot are detected and recorded via X-ray film or some other detection technique, thus providing a “profile” of the nucleotide. (Hybridization is used to pull out VNTR fragments, i.e. to separate them from irrelevant fragments.) In this approach, sample DNA is digested, and the resulting fragments are separated by size using gel electrophoresis. The separated fragments are transferred to a membrane by blotting, and are subjected to primer hybridization. (58)
This technique is time-consuming, labor intensive, and the gel may have a limited resolving power, making it potentially difficult to interpret the results. Another disadvantage is that these techniques generally require the use of a polymerase chain reaction (PCR) to multiply the polynucleotide in the sample. That is, the conventional tests are not very sensitive, and require relatively large DNA samples which often are not available. In such cases the sample concentration is increased to a meaningful detectable level by PCR. While this addresses some problems of sensitivity and sample degradation, PCR has been open to challenge because of possible sample contamination, and consequent undesirable amplification of contaminants leading to unreliable results. PCR approaches are also difficult to multiplex. For example, the probes and primers must be chosen with care, and generally only one set can be used. The sample may be consumed by one round of PCR, and different sets of probes or primers may require different reaction conditions, such as temperature. A simpler, more
Chou Hou-Pu
Quake Stephen R.
California Institute of Technology
Darby & Darby
Meyers Carla J.
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