332 MaundersDetection systems involve the use of an alkaline phosphatase mol-
ecule conjugated to a second protein that specifically recognizes the
target molecule. This protein may be a ligand, such as streptavidin,
which would bind to a biotin-labeled target, or an antibody that offers
more versatility. The presence of the target-detector-enzyme com-
plex is revealed by the action of alkaline phosphatase on a chromoge-
nic substrate (such as 5-bromo-4-chloro-3-indolylphosphate/Nitroblue
tetrazolium chloride, or BCIP/NBT), or a substrate that will produce
luminescence when dephosphorylated (e.g., 3-[2'-spiroadamantane]-
4-methoxy-4- [3-phosphoryloxy]-phenyl- 1,2-dioxetane, or AMPPD).
Such systems enable the detection of specific proteins by ELISA or
western blotting, or of DNA sequences either directly by detection of
haptens that have been linked to DNA probes (e.g., biotin, digoxygenin)
or indirectly by detection of the expression products of cloned genes.
Specific DNA molecules can also be detected by use of oligonucleotides
that have been conjugated directly to alkaline phosphatase (5). The
numerous variations and intricacies of these AP-linked systems are
beyond the scope of this chapter, which will now concentrate on the
direct action of alkaline phosphatases on phosphorylated nucleic acids.
The maj or application of alkaline phosphatase in molecular biology
is in dephosphorylating 5' termini of DNA or RNA to prevent self-
ligation. This is normally performed on vector DNAto reduce the number
of nonrecombinant molecules produced during cloning, but may also
be applied to the DNA to be cloned to prevent joining of small
noncontiguous fragments that would then give spurious products (6).
Dephosphorylation also enables subsequent tagging with radiola-
beled phosphate using the enzyme T4 polynucleotide kinase (see
Chapter 20). This approach can be used for DNA (7) or RNA (8)
sequencing, and for fragment mapping (9).
- The Enzyme
2.1. Bacterial Alkaline Phosphatase (BAP)
BAP is a dimer of identical or very similar subunits (10). The mol
mass has been determined as 67,000-110,000 Da, varying with pH and
ionic strength (11) owing in some measure to tetramer formation and
also transition to a random coil at low pH. The enzyme is often regarded
as a compact sphere of mol mass 80,000 Da (12). The sedimentation