Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-10-11
2003-05-27
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S912000, C536S023100, C536S024310, C536S023500, C536S024330
Reexamination Certificate
active
06569629
ABSTRACT:
BACKGROUND
Beef used for human consumption has a number of characteristics desired by consumers. Characteristics such as color, texture, firmness, tenderness and marbling all contribute to the quality of a cut of meat. Based on such characteristics, the United States Department of Agriculture (USDA) classifies beef from bulls, steers and heifers into eight different quality grades. Beginning with the highest and continuing to the lowest, the eight quality grades are prime, choice, select, standard, commercial, utility, cutter and canner. Typically, beef that is classified as prime or choice is sold at higher prices than beef that is classified into lower quality grades.
One particularly desired characteristic of beef is marbling which refers to the relative amount of intramuscular fat in the beef. Well-marbled beef, i.e., beef that contains substantial amounts of intramuscular fat relative to muscle, tends to be classified as prime or choice; whereas, beef that is not marbled tends to be classified as select. Another desirable characteristic of beef desired by consumers is tenderness which refers to the softness or the ease in chewing the meat, after it is cooked.
At present, marbling is determined after a bovine animal is slaughtered. For example, marbling of beef from carcasses is determined by a certified USDA grader at the packing facility and involves visual inspection of a region between the 12th and 13th rib of a beef carcass. Unfortunately, the visual appraisal by the grader is costly, labor intensive, and time-consuming.
At present, tenderness of beef is determined after it has been cooked. Two methods are used. The first involves a subjective analysis by a panel of trained testers. The second is the Warner-Bratzler shear force procedure which involves an instrumental measurement of the force required to shear steaks, chops, and ground patties of cooked beef. Both methods are costly, time-consuming and can only be used to determine tenderness after the animals have been harvested and the beef has been cooked.
It is desirable to have alternative methods to determine if the beef obtained from a carcass is marbled or tender. Methods which are inexpensive, rapid and require minimal labor are especially desirable.
Also, it is desirable to have methods for determining if live animals have the potential to provide beef that is well-marbled and tender. There are numerous advantages in determining characteristics, marbling and tenderness for example, of beef from a live animal. One advantage is that an animal can be channeled into a particular feeding regimen or used to meet requirements of specific marketing programs based on the marbling and tenderness characteristics of the beef from that animal.
Another advantage of determining characteristics of beef from live animals is that these characteristics can indicate a “genetic potential” possessed by the live animal to pass, for example, its marbling and tenderness characteristics on to the animal's offspring. For example, an animal with advantageous marbling or tenderness genetic potential can be bred with other animals containing one or more markers of marbling or tenderness for the purpose of developing inbred lines of animals whose beef is particularly marbled or tender. Such inbred lines would provide beef products with known and consistent marbling and tenderness characteristics. Unfortunately, there are a scarcity of methods for determining the characteristics of beef in live animals.
One recently developed method for determining both marbling and tenderness of beef, using samples from either live cattle or beef carcasses, is the method described in the commonly assigned U.S. Pat. No. 6,242,191 of Fluharty and Jackwood. This method comprises extracting DNA from a sample (e.g., blood) obtained from a bovine animal or beef carcass, amplifying the extracted DNA using specific primers under low stringency polymerase chain reaction (PCR) conditions to provide a pool of PCR products, and determining whether PCR products of specific sizes, which have been correlated with marbling or tenderness (i.e., marbling or tenderness markers), are present. The presence of the specific PCR products indicates that beef obtained from that animal or carcass will be marbled or tender, depending on which particular PCR products are present. The absence of the specific PCR products indicates that the beef obtained from that animal or carcass is not likely to be marbled or tender, also dependent on the particular PCR products present. This method, where PCR is performed under conditions where the specific primers bind to multiple specific locations within the extracted template DNA, such that a number of DNA sequences are amplified, is called a random amplified polymorphic DNA (RAPD) assay.
The RAPD assay is a vast improvement over earlier techniques. One advantage is that samples for testing can be obtained from live animals. Data obtained from such live-animal testing can be used to choose animals that should be interbred for the purpose of developing lines of bovine animals with the desired characteristics. Other advantages, in particular for testing of carcasses, is that the new method is objective and practical to perform on a large scale.
Although the RAPD assay is a major advance over what was previously available, it would be desirable to develop additional methods for testing beef. Such new methods would be specific, and possibly more easily adapted for use under various conditions. At the same time, it would be desirable to make these additional methods as simple to use as possible.
SUMMARY OF THE INVENTION
In accordance with the present invention, new methods are provided for objectively identifying: i) bovine animals having the genetic potential to produce beef that is marbled or tender, and ii) bovine carcasses whose beef is marbled or tender. The methods comprise extracting DNA from a sample obtained from a bovine animal or carcass, assaying for the presence of a DNA comprising a sequence, referred to hereinafter as a “genetic marker”, in the DNA sample. Preferably, the assay is a quantitative assay which is capable of determining the number of copies of the genetic marker in the DNA sample. In one aspect, the genetic marker is a marker of marbling, and comprises the sequence set forth in SEQ ID NO. 1. In another aspect, the genetic marker is a marker of tenderness, and comprises the sequence set forth in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or combinations thereof. In one embodiment the assay is a polymerase chain reaction (PCR) which employs primers that amplify all or a portion of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO. 4, and the complements thereof.
The present invention also provides DNA sequences whose presence in the genome of a bovine animal or carcass indicates that the animal has the genetic potential to produce marbled or tender beef, and that beef from the animal or carcass has the potential to be marbled or tender. These sequences can be used to provide: i) primers that amplify markers of marbled or tender beef present in bovine animal or carcass genomes, ii) hybridization probes to detect marbling or tenderness markers, and iii) probes to detect RNA transcribed from marbling or tenderness markers.
The present invention also provides oligonucleotides comprising sequences from DNA whose presence in an animal or carcass genome correlates with beef marbling or tenderness. These oligonucleotides can be used as hybridization probes, PCR primers and DNA sequencing primers to detect marbling or tenderness markers.
The present invention also provides a kit that can be used for analyzing samples from a bovine animal or carcass for the presence of marbling or tenderness markers. The kit comprises DNA sequences, primers and primers sets that can be used to assay for the presence of marbling and tenderness markers in the genomes of bovine animals or carcasses.
REFERENCES:
patent: 6242191 (2001-06-01), Fluharty et al.
patent: WO9953090 (1999-10-01), None
patent: WO0069882 (2000-11-01), None
“Development and
Fluharty Francis
Jackwood Daral J.
Calfee Halter & Griswold LLP
Myers Carla J.
The Ohio State University Research Foundation
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