Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-06-21
1998-11-03
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 911, 536 231, 536 254, C12Q 168, C12P 1934, C07H 2104
Patent
active
058306625
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND
Significance of cDNA Library Normalization
A typical somatic cell contains approximately 0.6 pg of mRNA. Thus, there are about 500,000 mRNA molecules per cell assuming that the average size of a mRNA is 2 kb (11.times.10.sup.7 pg). These mRNAs occur in three frequency classes (reviewed by Davidson and Britten, 1979):
______________________________________ # mRNA # copies Total
% mass species per species
mRNAs
______________________________________
Superprevalent
10 (10-20)
10 5,000 50,000
Moderately
45 (40-45)
1,000 225 225,000
Prevalent
Complex 45 (40-45)
15,000 15 225,000
______________________________________
frequency of 1/500,000. Its representation in a cDNA library will depend
on the number of independent recombinants. The probability that a given
mRNA will be represented can be expressed by the equation
P(x)=1-(1-f).sup.n, where f-frequency (1/500,000) and n-number of
recombinant clones. Therefore, the probability that the most rare mRNA
will not be represented in a cDNA library of 10.sup.7 recombinants is
2.times.10.sup.-9.
Although even the rarest mRNA will be represented in a library, its identification is very difficult (1/500,000). In a normalized cDNA library, however, the frequency of each clone is in the same narrow range and depends on the complexity of the library.
Assuming that there are 50,000 to 100,000 genes in the human genome (Bishop et al., 1974), an ideal normalized cDNA library from a great variety of tissues containing 1-2 kb cloned inserts of every single expressed human gene would have a complexity of 50,000 to 200,000 kb, and every clone would be represented at a frequency of 1/50,000 to 1/100,000, which would still be 5-10 times higher than the frequency of the most rare mRNA in a single somatic cell (1/500,000).
According to the considerations described above, the relative frequency of a member of each class of sequences (superprevalent, moderately prevalent and complex) in a representative cDNA library of a typical cell is I:II:III=1.7 and III=25. At Cot=250 (which is 10.times.the Cot.sub.1/2 of class III) of the leftover of each component, expressed as % of the initial amount, will be I=0.03%, II=0.6% and III=9%, while the relative average frequency of a member of each class will be 1:1:1, i.e., the library will be normalized.
Methods to Normalize cDNA Libraries
Thus far, two approaches have been proposed to obtain normalized cDNA libraries (Weissman, 1987). One approach is based on hybridization to genomic DNA. The frequency of each hybridized cDNA in the resulting normalized library would be proportional to that of each corresponding gene in the genomic DNA. The other is a kinetic approach. If cDNA re-annealing follows second-order kinetics, rarer species anneal less rapidly and the remaining single-stranded fraction of cDNA becomes progressively more normalized during the course of the hybridization (Galau, et al., 1977). Specific loss of any species of cDNA, regardless of its abundance, does not occur at any Cot value.
Two groups have pursued independently the construction of normalized cDNA libraries based on the kinetic approach (Ko, 1990; Patanjali et al., 1991).
Ko (1990) reported the construction of a normalized mouse cDNA library by a complex scheme involving: a) ligation of cDNAs to a linker-primer adapter; b) three rounds of PCR amplification, denaturation-reassociation, and purification of single-stranded cDNAs by hydroxyapatite (HAP) column chromatography; and c) digestion of the end product using a site present in the linker-primer sequence and cloning (#' non-coding cDNA fragments only) into a plasmid vector.
Colony hybridization with eight probes of different abundances showed a reduction in abundance variation from at least 20,000 fold in the original library to 40-fold in the library constructed after three cycles of normalization.
In Ko's method, both coding and non-coding fragments are present during reassociation. However, after the final digestion and directional cloning steps only the 3'
REFERENCES:
patent: 5482845 (1996-01-01), Soares et al.
patent: 5637685 (1997-06-01), Soares et al.
patent: 5702898 (1997-12-01), Bonaldo et al.
Ko, M.S.H., An `equalized cDNA library` by the reassociation of short doubled-stranded cDNAs, Nucleic Acids Research, 18(19): 5705-5711 (1990).
Patanjali, S.R., et al., Construction of a uniform-abundance (normalized) cDNA library, Proc. Natl. Acad. Sci. U.S.A. 88: 1943-1947 (1991).
NTIS Progress Report, Soares & Efstratiadis, Oct. 1992; and.
Sasaki, Yasnory F., et al., Construction of a normalized cDNA library by introduction of a semi-solid mRNA-cDNA hydridization system, Nucleic Acids Research, 22(6): 987-992, (1994).
Efstratiadis Argiris
Soares Marcelo B.
Horlick Kenneth R.
The Trustees of Columbia University in the City of New York
White John P.
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