22 Maunders- Klenow Fragment
3.1. Enzyme Data
By treatment of DNA polymerase I with the protease subtilisin, a
polypeptide of mol mass 35,000 Da is removed that carries the 5'-3'
exonuclease activity (6). The remaining moiety, of mol mass 76,000
Da, retains all the other enzymic activities and is commonly termed the
DNA polymerase I large fragment or Klenow fragment.
3.2. Uses of Klenow Fragment
It is impossible to use Klenow fragment for nick translation because
of the lack of a 5'-3' exonuclease activity. However, this limitation is
turned to advantage in other applications of the enzyme by rendering
the polymerization activity more controllable.
The primer extension activity of Klenow fragment has made it widely
used in dideoxynucleotide sequencing methods (7), whereby DNA
templates up to 350 bases long can be sequenced at a rate of 45 nucle-
otides/s. Several different DNA polymerases are commonly used for
sequencing reactions. Each has distinct abilities when encountering
particularly "difficult" regions of the template sequence.
The enzyme can also be used to produce labeled double-stranded
DNA probes (actually only labeled in one or the other of the two
strands) by the extension of oligomeric primers of random sequence
(8). The oligolabeling technique produces probes of a much higher
specific activity than nick translation, with over 55% of the labeled
nucleotide incorporated in 30 min at 37°C. Although the entire probe
sequence is represented in the labeling mix, individual molecules are
commonly only a few hundred bases in length, which may be a disad-
vantage for some applications. A typical reaction protocol is given in
Section 3.3.1.
Single-stranded (strand-specific) probes can also be prepared by
end-labeling of protruding, flush, or recessed 3' termini. This is achieved
by removal of the 3' nucleotides by means of the 3'-5' exonuclease
activity (which is more active on single-stranded than double-stranded
DNA), to form a recessed 3' end. Following the addition of high con-
centrations of deoxynucleotide triphosphates (one or more of which
may be labeled), the polymerase activity resynthesizes the excised
region (9,10). This process has advantages over nick translation in that