Page 355 - Applied Probability

Appendix A: Molecular Genetics in Brief
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A.3 Manipulating DNA
Geneticists manipulate DNA in many ways. For instance, they unzip (or
denature) double-stranded DNA by heating it in solution. They rezip
(or anneal) it by cooling. Because double helices are so energetically fa-
vored, complementary strands quickly find and bind to one another. Even
small segments of one strand will locally anneal to a large segment of a
complementary strand. Geneticists exploit this behavior by devising small
radioactive or fluorescent probes to identify large segments. A single base
mismatch between probe and strand leads to poor annealing. Probes as
short as 20 bases can provide a perfect match to a unique part of the
human genome.
TABLE A.3. Commonly Used Restriction Enzymes
Restriction Recognition Average Fragment
Enzyme Site Length in Man
AluI AGCT 0.3 kb
HaeIII GGCC 0.6 kb
TaqI TCGA 1.4 kb
HpaI CCGG 3.1 kb
EcoRI GAATTC 3.1 kb
PstI CTGCAG 7kb
NotI GCGGCCGC 9766 kb

Chromosomes are much too large to handle conveniently. To reduce DNA
to more manageable size, geneticists cut it into fragments and measure
the length of the fragments. Restriction enzymes function as geneti-
cists’ molecular scissors. Table A.3 lists some commonly used restriction
enzymes, each of which recognizes a specific base sequence and cuts DNA
there. Recognition sites are scattered more or less randomly throughout
the genome. Restriction maps characterize the number, order, and ap-
proximate separation of recognition sites on large DNA segments. These
maps are laborious to prepare and involve digesting a segment with differ-
ent combinations of restriction enzymes or with a single enzyme at less than
optimal laboratory conditions. These latter partial digests randomly miss
some recognition sites and therefore give a mixture of fragments defined
by adjacent sites and fragments spanning blocks of adjacent sites. Water-
man [9] discusses the interesting computational issues that arise in piecing
together a restriction map.
Gel electrophoresis and Southern blotting are geneticists’ molecular
yardsticks. In electrophoresis, a sample of DNA is placed at the top of
a gel subject to an electric field. Under the influence of the field, DNA
migrates down the gel. Large DNA fragments encounter more obstacles