Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
2001-08-09
2003-12-30
Zeman, Mary K. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C702S019000, C702S027000, C435S006120, C435S007100
Reexamination Certificate
active
06671625
ABSTRACT:
FIELD OF THE INVENTION
The invention generally relates to techniques for analyzing biological samples such as DNA, RNA, or protein samples and in particular to techniques for analyzing the output patterns of hybridized biochip microarrays.
BACKGROUND OF THE INVENTION
A variety of techniques have been developed to analyze DNA or other biological samples to identify diseases, mutations, or other conditions present within a patient providing the sample. Such techniques may determine, for example, whether the patient has any particular disease such as cancer or AIDS, or has a predisposition toward the disease, or other medical conditions present in the patient. DNA-based analysis may be used either as an in-vitro or as an in-vivo control mechanism to monitor progression of disease, assess effectiveness of therapy or be used to design dosage formulations. DNA-based analysis is used verify the presence or absence of expressed genes and polymorphisms.
One particularly promising technique for analyzing biological samples uses a DNA-based microarray (or microelectronics biochip) which generates a hybridization pattern representative of the characteristics of the DNA within the sample. Briefly, a DNA microarray includes a rectangular array of immobilized single stranded DNA fragments. Each element within the array includes few tens to millions of copies of identical single stranded strips of DNA containing specific sequences of nucleotide bases. Identical or different fragments of DNA may be provided at each different element of the array. In other words, location (
1
,
1
) contains a different single stranded fragment of DNA than location (
1
,
2
) which also differs from location (
1
,
3
) etc. Certain biochip designs may replicate the nucleotide sequence in multiple cells.
DNA-based microarrays deploy chemiluminiscence, fluorescence or electrical phenomenology to achieve the analysis. In methods that exploit fluorescence imaging, a target DNA sample to be analyzed is first separated into individual single stranded sequences and fragmented. Each sequence being tagged with a fluorescent marker molecule. The fragments are applied to the microarray where each fragment binds only with complementary DNA fragments already embedded on the microarray. Fragments which do not match any of the elements of the microarray simply do not bind at any of the sites of the microarray and are discarded during subsequent fluidic reactions. Thus, only those microarray locations containing fragments that bind complementary sequences within the target DNA sample will receive the fluorescent molecules. Typically, a fluorescent light source is then applied to the microarray to generate a fluorescent image identifying which elements of the microarray bind to the patient DNA sample and which do not. The image is then analyzed to determine which specific DNA fragments were contained within the original sample and to determine therefrom whether particular diseases, mutations or other conditions are present in the patient sample.
For example, a particular element of the microarray may be exposed to fragments of DNA representative of a particular type of cancer. If that element of the array fluoresces under fluorescent illumination, then the DNA of the sample contains the DNA sequence representative of that particular type of cancer. Hence, a conclusion can be drawn that the patient providing the sample either already has that particular type of cancer or is perhaps predisposed towards that cancer. As can be appreciated, by providing a wide variety of known DNA fragments on the microarray, the resulting fluorescent image can be analyzed to identify a wide range of conditions.
Unfortunately, under conventional techniques, the step of analyzing the fluorescent pattern to determine the nature of any conditions characterized by the DNA is expensive, time consuming, and somewhat unreliable for all but a few particular conditions or diseases. One major problem with many conventional techniques is that the techniques have poor repeatability. Hence, if the same sample is analyzed twice using two different chips, different results are often obtained. Also, the results may vary from lab to lab. Consistent results are achieved only when the target sample has high concentrations of oligonucleotides of interest. Also, skilled technicians are required to prepare DNA samples, implement the hybridization protocol, and analyze the DNA microarray output possibly resulting in high costs. One reason that repeatability is poor is that the signatures within the digitized hybridization pattern (also known as a “dot spectrogram”) that are representative of mutations of interest are typically very weak and are immersed in considerable noise. Conventional techniques are not particularly effective in extracting mutation signatures from dot spectrograms in low signal to noise circumstances. Circumstances wherein the signal to noise ratio is 0 to strongly negative (−2 to −30 dB) are particularly intractable.
Accordingly, it would highly desirable to provide an improved method and apparatus for analyzing the output of the DNA microarray to more expediently, reliably, and inexpensively determine the presence of any medical conditions or concerns within the patient providing the DNA sample. It is particularly desirable to provide a technique that can identify mutation signatures within dot spectrograms even in circumstance wherein the signal to noise ration is extremely low. It is to these ends that aspects of the invention are generally drawn.
Referring now to
FIG. 1
, conventional techniques for designing DNA microarray chips and for analyzing the output thereof will now be described in greater detail. Initially, at step
100
, fluorescently labeled primers are prepared for flanking loci of genes of interest within the DNA sample. The primers are applied to the DNA sample such that the fluorescently labeled primers flank genes of interest. At step
102
, the DNA sample is fragmented at the locations where the fluorescently labeled primers are attached to the genes of interest to thereby produce a set of DNA fragments, also called “oligonucleotides” for applying to the DNA microarray.
In general, there are two types of DNA microarrays: passive hybridization microarrays and active hybridization microarrays. Under passive hybridization, oligonucleotides characterizing the DNA sample are simply applied to the DNA microarray where they passively attach to complementary DNA fragments embedded on the array. With active hybridization, the DNA array is configured to externally enhance the interaction between the fragments of the DNA samples and the fragments embedded on the microarray using, for example, electronic techniques. Within
FIG. 1
, both passive hybridization and active hybridization steps are illustrated in parallel. It should be understood that, currently for any particular microarray, either the passive hybridization or the active hybridization steps, but not both, are typically employed. Referring first to passive hybridization, at step
104
a DNA microarray chip is prefabricated with oligonucleotides of interest embedded or otherwise attached to particular elements within the microarray. At step
106
, the oligonucleotides of the DNA sample generated at step
102
are applied to the microarray. Oligonucleotides within the sample that match any of the oligonucleotides embedded on the microarray passively bind with the oligonucleotides of the array while retaining their fluorescently labeled primers such that only those locations in the microarray having corresponding oligonucleotides within the sample receive the primers. It should be noted that each individual nucleotide base within the oligonucleotide sequence (with lengths ranging from 5 to 25 base pairs) can bond with up to four different nucleotides within the microarray, but only one oligonucleotide represents an exact match. When illuminated with fluorescent light, the exact matches fluoresces most effectively and the non-exact matches fluoresce considerably less or n
Kukkonen, III Carl A.
ViaLogy Corp.
Zeman Mary K.
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