Concatemeric DNA length standards

• US 5,294,545 A
• Filed: 07/03/1991
• Issued: 03/15/1994
• Est. Priority Date: 01/04/1989
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Serwer, Philip, Son, Marjatta
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): KETTER, JAMES S
• Application Number:
  US07/725,048
• Time in Patent Office: 986 Days
• Field of Search: 435/172.3, 435/6, 435/91, 435/320.1, 536/27, 536/2.1, 536/23.71, 204/182.2
• US Class Current: 435/91.53
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales
• CPC Class Codes: Y10S 435/849 Escherichia coli
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing

Abstract

The use of T7 bacteriophage to produce DNA length standards by enzymatically joining terminally repetitious, blunt-ended DNA has now been demonstrated. It is now possible to precisely control the formation of concatemeric DNAs thereby generating custom-made size-ranges of length standards. Furthermore, the standards thus produced are stable over time providing a highly reproducible and convenient product for the molecular biologist.

Claims

1. A method for sizing nucleic acid polymers of unknown length comprising the steps of:

a) loading into one well of a gel matrix a quantity of a concatemeric DNA standard prepared by

i) separately infecting at least two T7 infectable bacterial cell cultures each with a different T7 bacteriophage where the infecting bacteriophage is selected so that functional T7 gene 6 is present in at least one of the bacteriophage;

ii) allowing the cells to synthesize bacteriophage proteins;

iii) preparing extracts of the separately infected cells by lysing them in a buffered solution;

iv) mixing the extracts;

v) adding substrate DNA to the mixture and allowing enzymatic concatemerization of the substrate DNA to proceed for a selected time while limiting the packaging of the concatemeric DNA into bacteriophage head assemblies;

vi) stopping enzymatic concatemerization by inactivating the enzymes of the extract which mediate concatemerization of the DNA concatemers; and

,

vii) treating the concatemers with a protease to enable their use as a concatemeric DNA size standard;

b) loading into a separate well of the same gel matrix a quantity of a selected nucleic acid polymer;

c) electrophoresing the standard and the selected nucleic acid polymer through the gel matrix under conditions and for a time sufficient to resolve the concatemeric standard into a series of discrete bands where each band contains either monomeric substrate DNA or concatemers thereof; and

,

d) determining the size of the nucleic acid polymer by detecting its position in the gel after electrophoresis relative to the position of at least one of the bands of the concatemeric standard.

3. The method of claim 1 where the first bacterial cell culture is infected with T7 bacteriophage having mutations in genes 4 and 9 and the second bacterial cell culture is infected with a T7 bacteriophage having mutations in genes 5 and 19.

4. The method of claim 1 where the packaging of concatemeric DNA is limited by carrying out concatemerization at a temperature that is sufficiently low to eliminate packaging but high enough to allow concatemerization.

5. The method of claim 4 where the temperature is maintained between about 0°

-4°

C.

6. The method of claim 1 where the packaging of concatemeric DNA is limited by using two bacteriophage infected cell extracts devoid of viral gene products selected from the group consisting of 8, 9, 10, 18, 19 and some combination thereof such that the mixed cell extracts lack completely one or more of the gene products responsible for packaging.

7. The method of claim 6 where the packaging of concatemeric DNA is further limited by carrying out the concatemerization at a temperature that is sufficiently low to eliminate packaging but high enough to allow concatemerization.

10. The method of claim 1 or claim 2 where electrophoresis is accomplished by rotating the gel periodically and discontinuously in the presence of an electric field.

11. The method of claim 1 or claim 2 where the electrophoresis is accomplished using pulsed field gel electrophoresis.

12. The method of claim 1 or claim 2 where the buffered solution comprises a buffer of about pH 7.4 having the capacity to function at temperatures below 30°

C.

13. The method of claim 1 or claim 2 where the substrate DNA is obtained from T7 bacteriophage or a mutant thereof.

14. The method of claim 1 or claim 2 where the substrate DNA is obtained from a T7-related bacteriophage or a mutant thereof.

15. The method of claim 1 or claim 2 where the substrate DNA contains nonviral DNA.

16. The method of claim 1 or claim 2 where the substrate DNA contains synthetic DNA.

17. The method of claim 1 or claim 2 where inactivation of the enzymes of the extract is accomplished by addition of a buffer comprising 0.1M NaCl, 0.1M EDTA, 0.033M Tris-Hcl, pH 7.5, 1% Sarkosyl and 36 ug/ml RNase A.

18. The method of claim 1 or claim 2 where the protease is subtilisin.

19. The method of claim 1 or claim 2 further comprising embedding the concatemeric DNA in an agar block.

20. The method of claim 1 or claim 2 where all T7 bacteriophage used for infecting the bacterial cells are incapable of producing a functional product of T7 gene 3.

2. A method for sizing nucleic acid polymers of unknown length comprising the steps of:

a) loading into one well of a gel matrix a quantity of a concatemeric DNA standard prepared by

i) separately infecting at least two T7 infectable bacterial cell cultures each with a different T7 bacteriophage where (1) the infecting bacteriophage are selected so that functional T7 gene 6 is present in at least one of the bacteriophage, and where (2) one bacterial cell culture is infected with a first T7 bacteriophage characterized further in that the first bacteriophage contains mutations in at least one of the T7 genes responsible for viral DNA synthesis and in at least one of the T7 genes responsible for packaging of the viral DNA, and where (3) the other bacterial cell culture is infected with a second T7 bacteriophage characterized further in that the second bacteriophage contains a mutation in at least one of the T7 genes responsible for viral DNA synthesis distinct from that selected in the first bacteriophage and contains a mutation in the same T7 gene or genes responsible for packaging of the viral DNA as was mutated in the first bacteriophage;

ii) preparing extracts of the separately infected cells by lysing in a buffered solution where the buffer has a pH of about 7.4 and has the capacity to function below 30°

C.;

iii) mixing the extracts;

iv) adding substrate DNA to the mixture and allowing enzymatic concatemerization of the substrate DNA to proceed for a selected time while further limiting the packaging of the concatemeric DNA into bacteriophage head assemblies by carrying out the concatemerization at a temperature maintained at about 0°

-4°

C.;

v) stopping enzymatic concatemerization by inactivating the enzymes of the extract which mediate concatemerization of the DNA concatemers; and

,

vi) treating the concatemers with a protease to enable their use as a concatemeric DNA size standard; and

b) loading into a separate well of the same gel matrix a quantity of a selected nucleic acid polymer;

c) electrophoresing the standard and the selected nucleic acid polymer through the gel matrix under conditions and for a time sufficient to resolve the concatemeric standard into a series of discrete bands where each band contains either monomeric substrate DNA or concatemers thereof; and

,

d) determining the size of the nucleic acid polymer by detecting its position in the gel after electrophoresis relative to the position of at least one of the bands of the concatemeric standard.

8. The method of claim 2 where the gene responsible for DNA synthesis in the first bacteriophage is selected from the group consisting of genes 4 and 5 and the gene responsible for packaging in the first bacteriophage is selected from the group consisting of genes 8, 9, 10, 18 and 19.

9. The method of claim 2 where the gene responsible for DNA synthesis in the second bacteriophage is different from that selected in the first bacteriophage and is selected from the group consisting of genes 4 and 5 and the gene responsible for packaging in the second bacteriophage is identical to that selected in the first bacteriophage and is selected from the group consisting of gene 8, 9, 10, 18 and 19.