Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-29
2002-11-05
Arthur, Lisa B. (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06475729
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to very rapid amplification and detection methods to detect nucleic acids using polymerase chain reaction.
BACKGROUND OF THE INVENTION
Nucleic acid probe technology has developed rapidly in recent years as researchers have discovered its value for detection of various diseases, organisms or genetic features which are present in small quantities in a human or animal test sample. The use of probes is based upon the concept of complementarity. DNA has two strands bound together by hydrogen bonds between complementary nucleotides (which are also known as nucleotide pairs).
The DNA complex is normally stable, but the strands can be separated (or denatured) by conditions which disrupt the hydrogen bonding. The released single strands will reassociate only with another strand having a complementary sequence of nucleotides. This hybridization process can occur with both strands being in solution or with one of the strands being attached to a solid substrate.
A targeted nucleic acid sequence in an organism or cell may be only a very small portion of the entire DNA molecule so that it is very difficult to detect its presence using most labeled DNA probes. Much research has been carried out to find ways to detect only a few molecules of a targeted nucleic acid.
A significant advance in the art is described in U.S. Pat. No. 4,683,195 (issued Jul. 28, 1987 to Mullis et al), U.S. Pat. No. 4,683,202 (issued Jul. 28, 1987 to Mullis) and U.S. Pat. No. 4,965,188 (issued Oct. 23, 1990 to Mullis et al). Without going into extensive detail, these patents describe amplification and detection methods wherein primers are hybridized to the strands of a targeted nucleic acid (considered the templates) in the presence of a nucleotide polymerization agent (such as a DNA polymerase) and deoxyribonucleoside triphosphates. Under specified conditions, the result is the formation of primer extension products as nucleotides are added along the templates from the 3′-end of the primers. These products are then denatured and used as templates for more of the same primers in another extension reaction. When this cycle of denaturation, hybridization and primer extension is carried out a number of times (for example 25 to 30 cycles), the process which is known as “polymerase chain reaction” exponentially increases the original amount of targeted nucleic acid so that it is readily detected.
Once the targeted nucleic acid has been sufficiently amplified (that is, many times more copies of the molecule have been made), various detection procedures can be used to detect it. The patents noted above, for example, describe the use of insolubilized or detectably labeled probes and gel electrophoresis as representative detection methods.
In U.S. Pat. No. 4,965,188 (noted above), the cycle for amplification is generally described as follows:
a) denaturation at a temperature in the range of 90 to 105° C. (preferably 90 to 100° C.) for 0.5 to 5 minutes (preferably 0.5 to 3 minutes),
b) hybridization of primer to template at a temperature in the range of 35 to 65° C. (preferably 37 to 60° C.) for 0.5 to 5 minutes (preferably 1 to 3 minutes), and
c) formation of primer extension products at a temperature in the range of 40 to 80° (preferably 50 to 75° C.) for 0.5 to 40 minutes (preferably 1 to 3 minutes.
Thus, a wide range of times and temperatures are generally described with the specific combination of time and temperature largely dependent upon the type of DNA polymerase used, the complexity of the mixture of nucleic acids including the targeted nucleic acid, the length and specificity of the primers, the length of the targeted nucleic acid, pH and several other reaction conditions and components. There is no mention, however, of the time needed to change from one temperature to another, a factor which is largely dependent upon the type of heat transfer equipment used in the process. Thus, considerable effort must be carried out to find the optimum conditions for effective amplification and detection of a given nucleic acid.
One typical amplification cycle in Example II of U.S. Pat. No. 4,965,188 (noted above) requires about 5.5 minutes for a single cycle of the following steps:
a) heating the reaction mixture from 37 to 95° C. over three minutes,
b) denaturation of double strands at 95° C. for 0.5 minutes,
c) cooling to 37° C. over 1 minute, and
d) hybridization of primers to template and primer extension product formation at 37° C. for 1 minute.
Another typical amplification cycle is described in Example VII of U.S. Pat. No. 4,965,188, also requires 5.5 minutes and includes the steps:
a) heating the reaction mixture from 70 to 98° C. over 1 minute,
b) denaturing of double strands at 98° C. for 1 minute,
c) cooling to either 38, 45 or 55° C. over 1 minute,
d) hybridizing primers and template at 38, 45 or 55° C. for 1 minute,
e) heating from 38, 45 or 55° C. to 70° C. over 1 minute, and
f) forming primer extension products at 70° C. for 0.5 minute.
Since the discovery of the amplification and detection methods using polymerase chain reaction, there has been steady effort to find ways to carry out cycling in a rapid manner. A number of publications have suggested the desirability of fast cycling, but have not given suitable directions as to how it can be done. For example, rapid cycling is somewhat dependent upon suitable instrumentation. Details of such instrumentation are provided, for example, in EP-A-0 236 069 (published Sep. 9, 1987 and corresponding to U.S. Ser. No. 833,368 filed Feb. 25, 1986 and U.S. Ser. No. 899,061 filed Aug. 22, 1986). Cetus Corporation and Perkin-Elmer have developed commercially available thermocycling equipment which have enabled the user to perform a polymerase chain reaction cycle in from 3 to 6 minutes, similarly to the examples shown in U.S. Pat. No. 4,965,188 (noted above). While it may seem that a 3 to 6 minute cycle is quite fast, if one considers that efficient amplification generally requires 25 to 30 cycles to render the nucleic acid detectable, a typical standard amplification method could require 75 to 180 minutes.
More recently, Cetus Corporation and Perkin-Elmer have marketed a thermocycler (PCR System Model 9600) which allows the use of cycles of about 2 to 3 minutes in the amplification procedure.
Others have worked to find even faster cycling equipment which can be used with self-contained reaction vessels (sometimes known as cuvettes, pouches or test packs). Such equipment is described, for example in U.S. Ser. Nos. 452,666 and 452,932 (both filed Dec. 18, 1989 by Devaney Jr. et al), both as CIPs of U.S. Ser. No. 365,079 (filed Jun. 12, 1989). A typical reaction vessel useful with such equipment is described in U.S. Ser. No. 339,923 (filed Apr. 17, 1989 by Schnipelsky et al) as a CIP of U.S. Ser. No. 306,735 (filed Feb. 3, 1989).
It has been recognized that efficient heat transfer in the cycling of reaction mixtures will aid in the reduction of time required for a polymerase chain reaction cycle. For example, Wittwer et al (
Anal. Biochem
., 186 (2), pp. 328-331, May 1, 1990) describe the use of hot air for heat transfer with reaction mixtures. Cycles lasting 30, 60, 120 and 180 seconds are described with the following steps in each cycle (as described for the 30 second cycle with longer cycles having proportionately longer steps):
a) denaturation of double strands at 90-92° C. for 1 to 2 seconds,
b) cooling to 50 to 55° C. over 6 to 9 seconds,
c) hybridization at 50 to 55° C. for 1 to 2 seconds,
d) heating to 71 to 73° C. over 3 to 5 seconds, and
e) forming primer extension products for 5 to 10 seconds.
The advantages of hot air heat transfer are described by Wittwer et al, and it is speculated that amplification can occur in minutes depending upon the reaction vessel and heat transfer equipment. The particular equipment described by Wittwer et al has a number of practical disadvantages, however, including the difficulty in loading samples and reagents into very small diameter capillary tubes, the fragility of those tubes (sui
Backus John Wesley
Donish William Harold
Findley John Bruce
Sutherland John William H.
Arthur Lisa B.
Gowen Catherine K.
Johnson & Johnson Clinical Diagnostics Inc.
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