Reacción #1527017

Ecuación de reacción

deoxycytidine triphosphate

deoxyadenosine triphosphate

P-ATP

→

Tris·acetate

Condiciones de reacción

Tratamiento posterior

1. 1
   OtroThis produced a fragment with the sequence ##STR19## near the beginning of the rennin gene
2. 2
   ExtracciónAfter phenol extraction, ether extraction, and ethanol precipitation
3. 3
   workup.DISSOLUTIONthe DNA was redissolved in water
4. 4
   workup.ADDITIONtreated with S1 nuclease (Boehringer/Mannheim, 100 units) for 30 minutes at room temperature i a 50 μl reaction
5. 5
   workup.ADDITIONcontaining Buffer S
6. 6
   OtroThis enzyme removed the 5' single-stranded DNA from the fragment
7. 7
   Otroleaving the sequence ##STR20## Thus, the beginning of the rennin sequence
8. 8
   Otrofor 30 minutes
9. 9
   Otroat 37° C.
10. 10
    Otroin a 25 μl reaction
11. 11
    workup.ADDITIONcontaining Buffer Y
12. 12
    OtroThe reaction was terminated
13. 13
    workup.ADDITIONFour microliters of 10x ClaI buffer (1x=10 mM Tris·HCl pH 8, 10 mM MgCl2), and 10 μl of restriction endonuclease ClaI (Boehringer/Mannheim, 27 units) were added
14. 14
    OtroFour microliters were removed for analysis on a polyacrylamide gel
15. 15
    workup.ADDITIONfollowed by the addition of 1 μl of 10x ClaI buffer
16. 16
    workup.WAITThis mixture was incubated for an additional hour
17. 17
    OtroThe treated DNA containing the desired rennin sequences was purified by separation in a 2% agarose gel
18. 18
    workup.ADDITIONcontaining 40 mM Tris·acetate buffer pH 8.3
19. 19
    OtroThe DNA was visualized by long wave ultraviolet irradiation
20. 20
    Otroremoved from the gel by the freeze-thaw method

Procedimiento

A second treatment of the DNA with the Klenow fragment of E. coli DNA polymerase I was conducted as described above except that 0.05 mM deoxycytidine triphosphate was substituted for the 0.05 mM deoxyadenosine triphosphate of the previous reaction. This produced a fragment with the sequence ##STR19## near the beginning of the rennin gene. After phenol extraction, ether extraction, and ethanol precipitation, the DNA was redissolved in water and treated with S1 nuclease (Boehringer/Mannheim, 100 units) for 30 minutes at room temperature i a 50 μl reaction containing Buffer S. This enzyme removed the 5' single-stranded DNA from the fragment leaving the sequence ##STR20## Thus, the beginning of the rennin sequence is at the 5' end of the DNA fragment. A synthetic oligonucleotide containing a ClaI restriction site and ending with the nucleotides ATG (i.e. CATCGATG, Collaborative Research, Inc., 5 μg) was kinased with Y32 -P-ATP using T4 polynucleotide kinase ((P-L Biochemicals, 3 units) for 30 minutes at 37° C. in a 25 μl reaction containing Buffer Y. This kinased linker (about 200 pmoles was ligated to the treated DNA fragment (about 5 pmoles) by incubation with T4DNA Ligase (N.E. Biolabs, 900 units) at 15° C. overnight in Buffer L. The reaction was terminated by heating at 65° C. for 5 minutes. Four microliters of 10x ClaI buffer (1x=10 mM Tris·HCl pH 8, 10 mM MgCl2), and 10 μl of restriction endonuclease ClaI (Boehringer/Mannheim, 27 units) were added. The resulting mixture wax incubated at 37° C. for one hour. Four microliters were removed for analysis on a polyacrylamide gel, followed by the addition of 1 μl of 10x ClaI buffer, 10 μl of ClaI enzyme, and 3 μl water. This mixture was incubated for an additional hour. The treated DNA containing the desired rennin sequences was purified by separation in a 2% agarose gel containing 40 mM Tris·acetate buffer pH 8.3. The DNA was visualized by long wave ultraviolet irradiation and removed from the gel by the freeze-thaw method described above. This fragment was then ready for insertion into the appropriate vector.

Fuente