# US 4,396,713 A · Restriction endonuclease fingerprinting of kinetoplast DNA minicircles

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- Number: US 4,396,713 A
- Document ID: US4396713A
- Status: Granted
- Kind: A
- Application number: 06/206,200
- Filed: 1980-11-12
- Granted: 1983-08-02
- Earliest claimed: 1980-11-12
- Assignees: The Regents of the University of Calif.
- Inventors: Larry P. Simpson; Carlos M. Morel
- Web page: https://patentgrep.com/patent/US4396713A

## CPC

- C12Q 1/683: involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
- C12Q 1/6893: for protozoa
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- Y10S 435/803: Physical recovery methods, e.g. chromatography, grinding
- Y10S 435/81: Packaged device or kit
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Hemoflagellates are characterized by restriction endonuclease digestion of the mitochondrial DNA to provide for substantial cleavage of the kDNA network. The resulting electrophoretic profile of the digest can be used as a restriction fingerprint for distinguishing organisms and specific strains. The kDNA is found to be sufficiently stable through numerous passages of the organism to provide a reliable fingerprint.

## Claims

### Claim 1

A method for assaying for the presence in a sample of a stock or strain within a species of hemoflagellate which comprises:
isolating kDNA from a cell culture from said sample;
restriction endonuclease digesting said isolated kDNA with at least one restriction endonuclease selected from the group consisting of EcoRI, HaeIII, HinfI, MspI, and TaqI to provide for cleavage of minicircles present in said kDNA, wherein at least 20% of the minicircles are cleaved at least twice;
electrophoresing the digested kDNA under conditions of high resolution to provide a schizodeme profile unique to a particular hemoflagellate strain or stock; and
comparing said profile with a known hemoflagellate strain or stock digested and electrophoresed in substantially the same manner as said kDNA from said sample cell culture to determine the presence of said known hemoflagellate in said sample.

### Claim 2 (depends on 1)

A method according to claim 1, wherein said high resolution employs a linear gradient polyacrylamide gel.

### Claim 3 (depends on 1, 2)

A method according to claims 1 or 2, where one of said restriction enzymes is TaqI.

### Claim 4 (depends on 1)

A method according to claims 1, or 2, where one of said enzymes is EcoRI.

### Claim 5 (depends on 1)

A method according to claim 1, where said hemoflagellate is a trypanosome.

### Claim 6

A method for assaying for the presence in a sample of a Kinetoplastida strain, which comprises:
growing microorganisms in a nutrient medium from a sample suspected of containing Kinetoplastida;
lysing the cells and isolating the cells by means of centrifugation to provide a pellet;
suspending the pellet in an aqueous buffered medium and extracting said medium with at least one organic solvent;
digesting the kDNA with at least one restriction endonuclease selected from the group consisting of EcoRI, HaeIII, HinfI, MspI, and TaqI to cleave at least a substantial proportion of the minicircles present in said kDNA and at least 20% of said minicircles twice;
electrophoresing said digested kDNA under high resolution conditions to provide a profile characteristic of a particular Kinetoplastida strain; and
comparing said profile with a known hemoflagellate strain or stock digested and electrophoresed in substantially the same manner as said kDNA from said sample cell culture to determine the presence of said known hemoflagellate in said sample.

### Claim 7 (depends on 6)

A method according to claim 6, wherein said Kinetoplastida is a Trypanosoma.

### Claim 8 (depends on 6)

A method according to claim 6, wherein said Kinetoplastida is Leishmania.

### Claim 9 (depends on 6, 7, 8)

A method according to claims 6, 7 or 8, wherein said digestion employs TaqI.

### Claim 10 (depends on 9)

A method according to claim 9, wherein said electrophoresing employs a linear gradient polyacrylamide gel.

### Claim 11 (depends on 10)

A method according to claim 10, wherein said lysing is achieved with pronase in the presence of sarkosinate.