Linker scanning summary

Denis Tang DTANG at VM1.SI.USHERB.CA
Sat Dec 12 00:04:33 EST 1992



                                     Sherbrooke, 11 decembre 1992



          For those who read my message about "linker scanning", here is the
summary of the answers I've received:

From: CAMDNA at ubvms.cc.buffalo.edu (DN)

"Linker scanning is a technique for determining specific DNA sequences in a
region of interest that are important for some function. The technique is
also known as linker substitution analysis. Basically, a small linker
is substituted for wild-type sequence at various closely-spaced positions
across the region. In its "purest" form, linker scanning results in neither
insertion or deletion--only perfect substitution. Since a linker of (usually)
6-8 bp is used, the "resolution" of the analysis is slightly lower than that
of point mutations. However, since no gross spacing between sequences is
changed, LS is a higher resolution than deletion analysis.

As for methods, the classical way to generate linker substitutions is to
create deletions in a region from some external site, attach a linker to the
deleted end and sequence the result. When this is done from both directions,
and enough deletions have been made, specific deletions from either end
can be combined to create the substitution. A much faster method, which
requires more money in terms of material (but probably is more cost-effective
when time spent is taken into account), is to use site-directed mutagenesis.

Method 1:     --------(linker)AAGTAGATCGGATTC
              TCGATCGA(linker)-----------------
                             |
                            \/
                   Restriction digestion, ligation
                             |
                            \/
              TCGATCGA(linker)AAGTAGATCGGATTC

I believe the original description of the method was by Steve McKnight, used
to dissect the TK promoter. I have the reference somewhere, but not handy.
A more recent use is described in Mararhens and Stillman, Science (1992)
February 14 th issue."



From: IBELGAUFTS at genmic (Horst Ibelgaufts)

"... linker scanning is a special way of in vitro mutagenesis. it employs
deoxyribonuclease I which is used to linearise circular dna molecules
randomly. linkers are then introduced at the ends of the molecules which
are then recircularised. in this way one obtains a family of small
insertion mutations (the inserted linkers) whose localisation is easily
mapped because of the restriction site provided by the inserted linker.
By mapping different linker insertions randomly obtained you can get
a good overview of the biological functions of the dna regions in which
the linker insertions may have inactivated genes. the technique is
called linker scanning because you actually scan an entire dna region
by statistically introducing linker insertions.
As references try
Barany et al. DNA Prot. Eng. Tech 1> 29 1988
Barany et al. gene 37: 111/123 1985
Heffron, F. et al. PNAS 75: 6012 1978"


     Thanks to both of you for your answers.


                                        Denis Tang
                                        Dtang at udesvm
                                        Dtang at vm1.si.usherb,ca



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