Simultaneous isolation and quantitation of DNA

Details Simultaneous isolation and quantitation of DNA Simultaneous isolation and quantitation of DNA

1999-08-20

2004-01-06

Lacourciere, Karen (Department: 1635)

Chemistry: analytical and immunological testing

Involving an insoluble carrier for immobilizing immunochemicals

Carrier is inorganic

C436S527000, C435S006120, C435S091100, C423S335000, C536S025420

Reexamination Certificate

active

06673631

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to methods for isolating a defined quantity of a DNA target material from other substances in a medium to produce a suitable quantity of isolated DNA target material for further processing or analysis. The present invention particularly relates to methods for isolating a defined amount of DNA target material using a silica-containing solid support capable of reversibly binding a definable quantity of the DNA target material, such as magnetically responsive particles comprising silica or a silica derivative.
BACKGROUND OF THE INVENTION
Many analysis techniques which involve the testing of a DNA target material present in a particular medium only work well when the DNA target material is isolated from other material in the medium, and quantified after isolation therefrom. Isolation of a DNA target material from other components in a forensics sample (e.g. bodily fluids collected from a crime scene, blood or buccal cells collected from suspects, etc.) is critical to ensure that the other components present in the sample do not interfere with analysis of the DNA target material. Unfortunately, forensic samples are frequently so small or so degraded that quantitation of DNA target material isolated therefrom can be time consuming and difficult. Moreover, the variance between individuals in the amount of leukocytes present in a given volume of blood further increases the variance of the quantity of DNA isolated.
With the advent of DNA typing as a tool for paternity testing and for identification of biological samples present at crime scenes has come the need to develop reliable methods for isolating and quantifying small amounts of genomic DNA. In the United States, the need to develop such systems has come from Federal Bureau of Investigation establishment of a database of analytical results from thirteen short tandem repeat (“STR”) loci of human genomic DNA. These results are entered into a centralized database referred to as the Combined DNA Index System (“CODIS”). STR analysis systems are based upon the use of amplification reactions, which enable one to analyze very small amounts of DNA, even sub-nanogram amounts. However, amplification only works well when the amount of DNA to be amplified is within a defined range, and when it is substantially isolated from contaminants which can inhibit or interfere with the amplification reaction. Thus, before STR loci can be amplified and analyzed, the target DNA must be purified and quantitated to reduce the risk of observing amplification artifacts. Quantitation is important in other applications as well, such as DNA sequencing.
Procedures currently used to isolate and quantify genomic DNA for use in genetic identity typing are time consuming, and too disjointed to be amenable to automation. For example, the following procedure is typically used to isolate and quantify genomic DNA for amplification and analysis of STR loci, such as the CODIS loci. First, blood or buccal swabs are obtained from individuals using a variety of devices and volumes. Second, these samples are processed to isolate DNA of variable purity and integrity. Third, the DNA is quantitated for downstream procedures so that the appropriate amount can be used to avoid artifacts. Fourth, the DNA is amplified using reactions that include primers specific for each of the STR loci to be analyzed. Finally, the amplification products are analyzed on a gel or capillary electrophoresis system for genotype identification. For a commercial system designed for use in co-amplifying and analyzing all thirteen CODIS loci, see GenePrint® PowerPlex™ 1.1 and GenePrint® PowerPlex™ 2.1 systems (Promega Corporation, Madison, Wis.).
White blood cells are the primary source of DNA in the blood. There is considerable variability in the white blood cell content of blood, due either to variability between individuals or variability of samples from a given individual based on the health of the individual at the time the sample was obtained. A similar variability exists in buccal swab samples, compounded by variability in the type of swab used, and storage conditions of the sample before sample processing.
Both inside and outside the context of amplification of genomic DNA for DNA typing analysis, discussed above, with amplification via the polymerase chain reaction (PCR), too little template results in low band intensity or no resultant band amplification. Excess DNA template frequently results in overamplification. Overamplification is recognized by an excessive number of artifact peaks and stutter bands—defined as a minor peak directly below a major allele peak. There may also be a high incidence of background activity and “pull-up”, defined as the inability to separate the different color bands in a multiplex. Reamplification of a lesser quantity of DNA may be required if excessive artifacts are present. Stutter bands are particularly pronounced when excess DNA is present and capillary electrophoresis is used for the separation of PCR amplification products. Also, as with sequencing, the generation of full length amplification products can be inhibited when too much template DNA is present. In other words, in PCR amplification, excess template DNA can lead to the presence of partially amplified fragments and low amounts of completely amplified products.
More specifically, when PCR or other amplification methods are used in forensic applications to amplify DNA, when too much DNA is amplified in a single reaction, the sample is overamplified and the signal strength of the anticipated bands tends to fall outside the desired range of the detector. Traditionally, these difficulties are minimized by quantification of DNA after its purification, which requires additional steps, time and cost. In genetic identity testing, the presence of DNA in excess of that recommended for the analysis system employed often leads to uninterpretable results; this can waste very limited samples, particularly in the case of forensic analysis.
Another DNA application which requires accurate quantitation of the nucleic acid is sequencing. Sequencing of DNA is best done on samples of target DNA which have been isolated from other material present in a medium which can interfere with the sequencing reaction. It is also necessary to quantify samples of target DNA prior to initiation of a sequencing reaction. For example, in the area of DNA sequencing, the amount of DNA template in the sequencing reaction must be within a fairly narrow range. For example, when using plasmid DNA, 150-300 ng of DNA is recommended when using automated sequencing with BigDye™ Chemistry (Perkin Elmer Biosystems). When using PCR products as sequencing templates with the same sequencing system, 40-80 ng of DNA is recommended. Too much template may result in short sequence read length, poor resolution or higher error rates. With too little template, the signal strength is too weak for optimal sequence reading.
Plasmid DNA is typically a source of DNA for sequencing reactions. There is considerable variability in plasmid DNA content within a population of bacterial cultures due to such factors as variability in plasmid copy number per cell, variability in growth media used, and concentration of cell mass.
There are a variety of methods currently used to quantitate a DNA target material in a sample. One such method is spectrophotometric determination. In this method, absorbance readings of a sample of unknown concentration are taken at the wavelength corresponding to the maximum absorbance of the DNA target material. For example, absorbance at 260 nanometers (nm) (“A
260
”) is used to determine the concentration of DNA in a solution, while absorbance at 280 nm (“A
280
”)is used to determine the concentration of protein in a solution. An absorbance reading at 260 nm of 1 corresponds to about 50 micrograms (50 &mgr;g) per milliliter (&mgr;g/ml) for double-stranded DNA, 40 &mgr;g/ml for single-stranded DNA and RNA, and about 20 &mgr;g/ml for single-stranded oligonucleotides. The ratio between the readi

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