John M. S. Bartlett.pdf - Bio-Nica.info

Table 1
Examples of Various Applications and Methods for the Ligase Chain Reaction
References Barany (1) Jurinke (2) Khanna (5) Abravaya (38) Day (7) Reyes (8)
Wilson (37) Luo (3)
Zirvi (6)
Ligase Taq 15 U Pfu 4 U Tth 25 fmol Tth 5000 U Tth 10 U Amplicase 1.5 U
Buffers 20 mM/7.6 20 mM/7.5 20 mM/7.6 10 mM/7.5 20 mM/8.3
Tris
EPPS 50 mM/7.8
KCl 100 mM 20 mM 100 mM 20 mM 50 mM 25 mM
MgCl 2 10 mM 10 mM 10 mM 30 mM 10 mM 10 mM
NaCl 400 mM
EDTA
1 mM
NAD+ 10 mM 1 mM 10 µm 0.5 mM
DTT 10 mM 1 mM 10 mM
NP-40 0.10%
Triton X-100 0.01%
Primers 40 fmol 3.3 pmol 500 fmol (38) 30 nm 600 fmol 3.6 nm
Template 1 fmol (1); 0.74 fmol 500 fmol (3); 100 molecules 10–100 ng 250 ng
6–60 µg (37) 100 fmol (5); in 500 ng genomic
50 fmol (6) DNA
Conditions
Denaturation 94°C, 1 min 92°C, 20 sec 94°C, 15 sec 85°C, 30 sec 95°C, 15 sec 91°C, 30 sec
Lgation 64°C, 4 min 60°C, 40 sec 65°C, 4 min 60°C, 30 sec 65°C, 4 min 55°C, 6 min
No. cycles 20–30 25 20 25 5 (LDR) a 27
Target Sickle cell mutation E. coli lacI Human eIF-4E (3); HIV AZT-resistant Human gene Sickle cell
(1); p53, Ha-ras (37) K-ras (5); human mutants; Chlamydia CYP21 mutation
microsatellites (6) cryptic plasmid
Differential Size-labeled primers Size-labeled Fluorescent-labeled Microparticles Size-labeled Arbitrary
detection (1); 32 P label (37) primers; primers; coated with primers; sequence on
matrix-assisted microparticles anti-hapten; sandwich fluorescent primer for
laser coated with immunoassay labeled capture
desorption/ anti-hapten performed primers; detection hybridization;
ionization using the Abbott in Perkin–Elmer biotinylated
time-of- IMx automated GeneScan primer
flight-mass analyzer sequencing system streptavidinspectrometry
alkaline
phosphatase
conjugate
Ligase Chain Reaction 141
a Ligase detection reaction uses ligase chain reaction to detect previously amplified product.