Problem Statement: Consider the following DNA sequence (from firefly luciferase). Draw the sequencing gel pattern that forms as a result of sequencing the following template DNA with ddNTP as the capper.
atgaccatgattacg...
Solution:
Given DNA template: 5'-atgaccatgattacg...-3'
DNA synthesized: 3'-tactggtactaatgc...-5'
+-------------------------+
Gel pattern: lane ddATP | W | | || |
lane ddTTP | W | | | | | |
lane ddCTP | W | | | |
lane ddGTP | W || | |
+-------------------------+
- --> Electric Field --> +
Decreasing size -->
where "W" indicates the well position, and "|" denotes the DNA
bands on the sequencing gel.
Specifically, the possible oligonucleotides are:
--------------------------------------------
Treatment with Capped Oligo Size
ddNTP (bases)
--------------------------------------------
ddATP: actggtactaatgc...-5' 14
actaatgc...-5' 8
aatgc...-5' 5
atgc...-5' 4
ddTTP: tactggtactaatgc...-5' 15
tggtactaatgc...-5' 12
tactaatgc...-5' 9
taatgc...-5' 6
tgc...-5' 3
ddCTP: ctggtactaatgc...-5' 13
ctaatgc...-5' 7
c...-5' 1
ddGTP: ggtactaatgc...-5' 11
gtactaatgc...-5' 10
gc...-5' 2
--------------------------------------------
If we label each capper with a different fluorescent color (say, yellow Y, red R, green G, and blue B), we can simply conduct sequence reaction in one vial and inject the reaction mixture in a single lane in (flat or capillary) gel electrophoresis.
Specifically, the possible oligonucleotides are:
--------------------------------------------
Treatment with Capped Oligo Size
ddNTP (bases)
--------------------------------------------
Y-ddATP: Y-actggtactaatgc...-5' 14
Y-actaatgc...-5' 8
Y-aatgc...-5' 5
Y-atgc...-5' 4
R-ddTTP: R-tactggtactaatgc...-5' 15
R-tggtactaatgc...-5' 12
R-tactaatgc...-5' 9
R-taatgc...-5' 6
R-tgc...-5' 3
G-ddCTP: G-ctggtactaatgc...-5' 13
G-ctaatgc...-5' 7
G-c...-5' 1
B-ddGTP: B-ggtactaatgc...-5' 11
B-gtactaatgc...-5' 10
B-gc...-5' 2
--------------------------------------------
Gel eletrophoresis orders the above colored mixture based on size:
--------------------------------------------
Capped Oligo Size
(bases)
--------------------------------------------
R-tactggtactaatgc...-5' 15
Y-actggtactaatgc...-5' 14
G-ctggtactaatgc...-5' 13
R-tggtactaatgc...-5' 12
B-ggtactaatgc...-5' 11
B-gtactaatgc...-5' 10
R-tactaatgc...-5' 9
Y-actaatgc...-5' 8
G-ctaatgc...-5' 7
R-taatgc...-5' 6
Y-aatgc...-5' 5
Y-atgc...-5' 4
R-tgc...-5' 3
B-gc...-5' 2
G-c...-5' 1
--------------------------------------------
Given DNA template: 5'-atgaccatgattacg...-3'
DNA synthesized: 3'-tactggtactaatgc...-5'
+-------------------------+
Gel pattern: lane ddATP | W ||||||||||||||| |
+-------------------------+
- --> Electric Field --> +
Decreasing size -->
where "W" indicates the well/inject position, and "|" denotes
the DNA bands on the sequencing gel.
|
