Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-03-02
2004-06-15
Zeman, Mary K. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C702S019000, C702S020000
Reexamination Certificate
active
06750011
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains to a process which can be fully automated for accurately determining the alleles of STR genetic markers. More specifically, the present invention is related to performing PCR amplification on DNA, assaying the PCR products, and then determining the genotype of the PCR products. The invention also pertains to systems which can effectively use this genotyping information.
BACKGROUND OF THE INVENTION
To study polymorphisms in genomes, reliable allele determination of genetic markers is required for accurate genotyping. A genetic marker corresponds to a relatively unique location on a genome, with normal mammalian individuals having two (possibly identical) alleles
104
for a marker on an autosomal chromosome
102
, referring to FIG.
1
A. (Though there are other cases of 0, 1, or many alleles that this invention. addresses, this if characterization suffices for the background introduction.). One important class of markers is the CA-repeat loci. This class is abundantly represented throughout the genomes of many species, including humans.
A CA-repeat marker allele is comprised of a nucleic acid word
106
PQRST,
where P is the left PCR primer, T defines the right PCR primer, Q and S are relatively fixed sequences, and the primary variation occurs in the sequence R, which is a tandemly repeated sequence
108
of the dinucleotide CA, i.e.,
R=(CA)
n
,
where is n is an integer that generally ranges between ten and fifty. Thus, the length of the allele sequence uniquely determines the content of the sequence, since the only polymorphism is in the length of R.
One can therefore obtain genomic DNA, perform PCR amplification of a CA-repeat genetic marker location, and then assay the length of the allele sequences by differential sizing, typically done by differential migration of DNA molecules using gel electrophoresis. The resulting gel
110
should, in principle, fib clearly show the alleles of marker for each individual's genome. Further, these sizes can be determined quantitatively by reference to molecular weight markers
112
.
However, the PCR amplification of a CA-repeat location produces an artifact, often termed “PCR stutter”. Most likely due to slippage of the polymerase molecule on the nucleic acid polymer go in the highly repetitive CA-repeat region, the result is that PCR products are produced that correspond to deletions of tandem CA molecules in the repeat region. Thus, instead of a single band on a gel corresponding to the one molecule
PQ(CA)
n
ST,
an entire population of different size bands
{PQ(CA)
n
ST, PQ(CA)
n−1
ST, PQ(CA)
n−2
ST, . . . }
in varying concentrations is observed. This PCR stuttering
114
can be viewed as a spatial pattern p(x), or, alternatively, as a response function r(t) of an impulse signal corresponding to the assayed allele.
The stutter artifact can be extremely problematic when the two alleles of an autosomal CA-repeat marker are close in size. Then, their two stutter patterns overlap, producing a complex signal
116
. In the presence of background measurement noise, this complexity often precludes unambiguous determination of the two alleles. To date, this has prevented reliable automated (or even manual) genotyping of CA-repeat markers from differential sizing assays.
This overlap of stutter patterns can be modeled as a superposition of two corrupted signals. Importantly, (1) the corrupting response function is roughly identical for two closely sized alleles of the same CA-repeat marker, and (2) this response function is largely determined by the specific CA-repeat marker, the PCR conditions, and possibly the relative size of the allele. Thus, the response functions
114
can be assayed separately from the genotyping experiment
116
. By combining
118
the corrupted signal together with the determined response functions of the CA-repeat marker, the true uncorrupted allele sizes can be determined, and reliable genotyping can be performed.
A primary goal of the NIH/DOE Human Genome Project during its initial 5 year phase of operation was to develop a genetic map of humans with markers spaced 2 to 5 cM apart (E. P. Hoffman, “The Human Genome Project: Current and future impact,”
Am. J. Hum. Genet
., vol. 54, pp. 129-136, 1994), incorporated by reference. This task has already been largely accomplished in half the time anticipated, with markers that are far more informative than originally hoped for. In these new genetic maps, restriction fragment length polymorphism (RFLP) loci have been entirely replaced by CA repeat loci (dinucleotide repeats, also termed “microsatellites”) (J. Weber and P. May, “Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction,”
Am J Hum Genet
, vol. 44, pp. 388-396, 1989; J. Weber, “Length Polymorphisms in dC-dA . . . dG-dT Sequences,” Marshfield Clinic, Marshfield, Wis., assignee code 354770, U.S. Pat. #5,075,217, 1991), incorporated by reference, and other short tandem repeat markers (STRs). It is expected that at least 30,000 CA-repeat markers will be made available in public databases in the form of PCR primer sequences and reaction conditions. One of the advantages of CA repeat loci is their high density in the genome, with about 1 informative CA repeat every 50,000 bp: this permits a theoretical density of approximately 20 loci per centimorgan. Another advantage of CA repeat polymorphisms is their informativeness, with most loci in common use having PIC values of over 0.70 (J. Weissenbach, c. Gyapay, C. Dib, A. Vignal, J. Morissette, P. Millasseau, G. Vaysseix, and M. Lathrop, “A second generation linkage map of the human genome,”
Nature
, vol. 359, pp. 794-801, 1992; G. Gyapay, et. al.,
Nature Genetics
, vol. 7, pp. 246-239, 1994), incorporated by reference. Finally, these markers 43; are PCR-based, permitting rapid genotyping using minute quantities of input genomic DNA. Taken together, these advantages have facilitated linkage studies by orders of magnitude: a single full-time scientist can cover the entire genome at a 10 cM resolution and map a disease gene in an autosomal dominant disease family in about 1 year (D. A. Stephan, N. R. M. Buist, A. B. Chittenden, K. Ricker, J. Zhou, and E. P. Hoffman, “A rippling muscle disease gene is localized to 1q41: evidence for multiple genes,”
Neurology
, in press, 1994), incorporated by reference.
The CA repeat-based genetic maps are not without disadvantages. First, alleles are detected by size differences in PCR products, which often differ by as little as 2 bp in a 300 bp PCR product. Thus, these alleles must be distinguished using high resolution sequencing gels, which are more labor intensive and technically demanding to use than most other electrophoresis systems. Second, referring to
FIG. 2
, CA repeat loci often show secondary “stutter” or “shadow” bands in addition to the band corresponding to the primary allele, thereby complicating allele interpretation. These stutter bands may be due to errors in Taq polymerase replication during PCR, secondary structure in PCR products, or somatic mosaicism for allele size in a patient. Allele interpretation is further complicated by the differential mobility of the two complementary DNA strands of the PCR products when both are labelled. Finally, sequencing gels often show inconsistencies in mobility of DNA fragments, making it difficult to compare alleles of individuals between gels and often within a single gel. The most common experimental approach used for typing CA repeat alleles involves incorporation of radioactive nucleotide precursors into both strands of the PCR product. The combined consequence of stutter peaks and visualization of both strands of alleles differing by 2 bp often leads to considerable “noise” on the resulting autoradiograph “signals”, referring to
FIG. 2
, which then requires careful subjective interpretation by an experienced scientist in order to determine the true underlying two alleles.
The stuttered signals of di-, tri-, tetra-, and other polynucleotide
Schwartz Ansel M.
Zeman Mary K.
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