Assay methods for nucleic acid in a sample

Details Assay methods for nucleic acid in a sample Assay methods for nucleic acid in a sample

1998-02-13

2003-10-21

Siew, Jeffrey (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S091520, C435S091510, C536S025320, C536S025600

Reexamination Certificate

active

06635418

ABSTRACT:

FIELD OF INVENTION
The present invention relates to a method for the determination of the presence and amount of DNA in a sample. The method is based on the use of a nucleic acid template dependent enzyme in combination with a random primer to generate an enzymatic product which incorporates a binding species and a detectable species covalently linked.
BACKGROUND OF INVENTION
The development of therapeutic biopharmaceuticals for human injection generated through recombinant deoxyribonucleic acid (DNA) technology has resulted in new standards for product purity. Viral contamination of several vaccines initially demonstrated the safety risk associated with the products generated from recombinant technology. Thus, contamination of products by host cell DNA could be a biological hazard for the recipient. The primary concern of regulatory agencies is the potential contamination of product with oncogenes or infectious viral DNA. DNA in high amounts (1-10 &mgr;g) has been shown to cause tumors in mice. Because the risk associated with exposure of 100 pg of DNA per dose is negligible, the WHO (World Health Organization) currently requires the DNA in all biopharmaceuticals to be below 100 pg per dose. Thus, methodologies that accurately determine picogram amounts of DNA have become a requisite in the biopharmaceutical field. Several different methodologies exist for quantitation of DNA. Each method has limitations with respect to dynamic range for accurate DNA quantitation and biases due to base composition. Because of the multitude of manipulations during the purification process of a typical protein therapeutic sample, the DNA in the final protein therapeutic is most likely sheared (less than 1,000 basepairs) and exists in both single and/or double stranded forms.
1. Current Methods for Determination of DNA
The most widely accepted method for quantitation of DNA is absorbance spectroscopy. At 260 nm, each base has a specific absorption spectra. Using a spectrophotometer, an average absorbance is obtained which directly reflects the base composition of the DNA. This reading is based on the base composition of the DNA because at an OD of 260, the extinction coefficient of adenosine and guanosine bases is more than cytosine or thymine. The discrimination on base composition and the low level of sensitivity (1 ug DNA) are several limitations of this method. Another significant drawback of this method is the interference caused by impurities such as organic compounds and protein.
A second method for quantitation of DNA utilizes fluorescence spectroscopy.
Upon binding DNA, the fluorescence characteristics of flourochromes change such that energy emitted at a particular wavelength is proportional to the amount of DNA in the sample. Typically, unknowns are extrapolated from a standard curve of known calibrated DNA standards. Several dyes currently exist that have the capacity to bind single or double stranded DNA, as well as mRNA. Ethidium bromide is one dye that binds to DNA without base discrimination and can be used to accurately quantitate at least 100 ng of DNA. Although the Hoechst dye (33258, Molecular Probes Eugene, Oreg.) quantitates 10 ng of DNA accurately (DyNA Quant; Hoefer Pharmacia Biotech), it preferentially binds AT rich regions of DNA. The Beacon DNA dye (PanVera Corp., Madison, Wis.) detects 25 ng/ml of double stranded DNA only. Molecular Probes manufactures two dyes that the company claims to accurately detect 25 pg/ml of double stranded DNA (PicoGreen) and 100 pg/ml of single stranded DNA (OliGreen). If both forms of DNA are represented in a given sample, two separate determinations must be performed. A PCR based method exists that amplifies the DNA and measures the relative amounts of DNA by changes in fluorescence polarization. All these fluorochromes have limitations with respect to the ability to detect low picogram amounts of DNA.
Another method for quantitation of DNA utilizes a specific labeled probe to quantitate a unique sequence of host DNA. Currently, the Southern slot-blot method is accepted by the FDA for the final determination of total DNA in protein therapeutic samples. Specifically, a labeled probe generated from purified host DNA is used to quantitate the presence of specific sequences of DNA in the final protein therapeutic sample. Although slot-blot detects low picogram quantities of DNA, the results represent only the host DNA. Therefore, contaminating DNA from other genes of the host or from other contaminants (such as bacterial DNA) are not detected. Major disadvantages of slot-blot include a relatively narrow dynamic range, a technically challenging protocol, a time consuming, and difficult protocol to troubleshoot. Additionally, the semi-quantitative results obtained from this method are directly dependent on the specific activity of the probe which can be highly variable.
Another commonly used methodology (for quantitation of 2-200 picograms of DNA) is an assay that captures DNA by binding to a single stranded DNA binding protein and detects the DNA via a enzymatic amplification of signal from a enzyme labeled anti-DNA antibody. Disadvantages to the total DNA assay performed on a threshold system (Molecular Devices) include the narrow dynamic range, the technically challenging and time consuming protocol. In addition the inability to accurately detect fragments of DNA below 872 basepairs severely limits the overall sensitivity of the assay. In fact, decreasing the size of DNA below 872 basepairs inhibits the detection of fragments greater than 872 basepairs. The lack of sensitivity for smaller DNA fragments is a major disadvantage of this methodology because the significant quantity of DNA in protein therapeutic samples is fragmented.
Chemical modification of DNA bases followed by detection via an enzyme labeled antibody that recognizes specific modified base is another technique that claims to detect pg amounts of DNA in samples. However, this method is complicated, extremely technically challenging, requires handing and requires subsequent disposal of hazardous waste, and extensive data manipulation.
2. Current Technology Using Random Oligonucleotide Priming
DNA dependent DNA polymerases incorporate deoxyribonucleotide to the 3′ hydroxyl terminus of a double stranded primed DNA molecule. The synthesis of the new strand of DNA occurs in a 5′ to 3′ direction with respect to the synthesized strand. Each nucleotide that is incorporated during the polymerization is complementary to the one opposite to it in the template(dA pairs with dT, dC with dG). The reaction requires four deoxyribonucleotide triphosphates (dNTPs) and magnesium ions. Many of the polymerases have a 3′-5′ exonuclease inherently associated with the polymerase activity. During DNA synthesis, the exonuclease activity performs a proofreading function by removing mismatched and modified nucleotides. One property of DNA polymerases that becomes important for the random priming reactions is the ability to continuously incorporate nucleotides without dissociating from the primer template (processivity). The low processivity associated with the klenow fragment (C-terminus) of DNA polymerase allows incorporation of less than 10 nucleotides into the primer template and thus maximizes the generation of signal. An outline of the properties of some polynucleotide dependent polymerases are provided in the table below.
3′-5′
Enzyme
exonuclease
Polymerase rate
Processivity
klenow fragment
low
intermediate
low
Reverse transcriptase
none
slow
intermediate
Taq DNA polymerase
none
fast
high
Several kits have been generated based on the properties of these polymerases. These protocols have become widely acceptable methodologies for labeling DNA and generating probes. Included in these methods are nick-translation of double stranded DNA and random priming of DNA using hexamer oligonucleotides. Both these protocols utilize DNA polymerases to incorporate the signal moiety (labeled-dNTP) into the DNA. In addition to DNA polymerase, terminal transferase can also be

Affiliated with

Also associated with

Rating

Assay methods for nucleic acid in a sample does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Assay methods for nucleic acid in a sample, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Assay methods for nucleic acid in a sample will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3171593