 |
|
|
Cleavage Close to the End of DNA Fragments (oligonucleotides)
To test the varying requirements restriction endonucleases have for the number of bases flanking their recognition sequences, series of double-stranded oligonucleotides that contain the restriction endonucleases recognition sites were digested. In the table shown only one strand of full complementary blunt-ends duplex, recognition sites marked as red.
The experiment was performed as follows: each strand of duplex was phosphorylated using T4 Polynucleotide Kinase and gamma-[32P]ATP. 4 10-4 A260 units of 5'[32P]-labeled oligonucleotide and same quantity of complementary non-labeled oligonucleotide was incubated with 1 mcl of restriction endonuclease at optimal for each enzyme conditions (temperature and buffer). Aliquots were taken at 2 hours and 20 hours and analyzed by 20%PAGE (7M urea). Percent cleavage was determined using Packard Cyclon Storage Phosphor System and computer program OptiQuant Version 3.0.
For each duplex was performed two experiments: in case of "Phosphorylated oligonucleotides" labeled oligonucleotide was phosphorylated at the end which is nearest to recognition site; in case of "Non-Phosphorylated oligonucleotides" labeled oligonucleotide was phosphorylated at the end which is remote from recognition site. |
Phosphorylated oligonucleotides
|
| Enzyme |
Oligonucleotide sequence |
Chain Length |
Cleavage, % |
| 2 h |
20 h |
| PciI |
TACATGTGTTAACAGCGCTA
AGTACATGTGGGCCCAGTACTGCGCAGCGCTAAC |
20
34 |
0
50 |
0
75 |
| AfeI |
TAGCGCTGTTAACACATGTA
GTTAGCGCTGCGCAGTACTGGGCCCACATGTACT |
20
34 |
0
50 |
0
50 |
| PsiI |
CTTATAAGTGCACGGGCCCA
AGTTTATAACTGGGCCCAAC |
20
20 |
25
>90 |
50
>90 |
| PspOMI |
TGGGCCCGTGCACTTATAAG
GTTGGGCCCAGTTATAAACT |
20
20 |
>90
>90 |
>90
>90 |
| BstAPI |
AGCAAATATTGCTCGATATC
ACTGCAGGCCTTGCCATATG
ACTGAAGCAATATTTGCCGATTA |
20
20
23 |
>90
>90
>90 |
>90
>90
>90 |
Non-Phosphorylated oligonucleotides
|
| Enzyme |
Oligonucleotide sequence |
Chain Length |
Cleavage, % |
| 2 h |
20 h |
| PciI |
TAGCGCTGTTAACACATGTA
GTTAGCGCTGCGCAGTACTGGGCCCACATGTACT |
20
34 |
25
25 |
25
50 |
| AfeI |
TACATGTGTTAACAGCGCTA
AGTACATGTGGGCCCAGTACTGCGCAGCGCTAAC |
20
34 |
0
50 |
0
50 |
| PsiI |
TGGGCCCGTGCACTTATAAG
GTTGGGCCCAGTTATAAACT |
20
20 |
75
>90 |
75
>90 |
| PspOMI |
CTTATAAGTGCACGGGCCCA
AGTTTATAACTGGGCCCAAC |
20
20 |
25
>90 |
>90
>90 |
| BstAPI |
GATATCGAGCAATATTTGCT
CATATGGCAAGGCCTGCAGT
TAATCGGCAAATATTGCTTCAGT |
20
20
23 |
>90
>90
>90 |
>90
>90
>90 |
| |
