Chemistry Reference
In-Depth Information
organizational information, for identifying specific genes that code for proteins, for
assaying the transcriptional and translational processes, and for identifying cells which
express certain genes. One technique is
restriction endonuclease fragmentation
, which
allows us to cut dsDNA into specific sized lengths for analyses (Fig. 8). This is
accomplished by the use of bacterial DNA cutting enzymes (restriction endonucleases)
that recognize specific base pair sequences within double stranded DNA and cut specific
phosphodiester bonds within both strands, leaving either cut, flush-ended double stranded
DNA, or double stranded DNA with short, single stranded ends (i.e., “sticky ends”).
Figure 8 shows an example of each type of enzyme (Bam H1, Hae III); note that the
name of each enzyme is derived from the abbreviated name of the parent bacterial strain.
Restriction endonucleases are used to cleave dsDNA molecules into specific fragments
that are more readily analyzed and manipulated. By the use of multiple restriction
endonucleases, we can generate a fragment map or “fingerprint” of a specific DNA.
Among other things, these enzymes allow us to excise a specific gene region and splice
or insert a different one in its place, or, to obtain a specific DNA fragment that can be
used to screen other genomic or RNA libraries.
A technique which complements restriction fragmentation is
DNA ligation
. A DNA
enzyme called DNA ligase catalyzes the formation of a phosphodiester bond between
dsDNA molecules containing free 3ƍ
OH and 5ƍ phosphate groups. (Fig. 8). Ligation can
occur between flush-ended DNA molecules, or between DNA molecules that possess
“sticky ends.” This technique allows the joining of different DNA fragments together to
form new DNA molecules.
We also have the ability to “tag” or label any piece of DNA, such that it can be
identified and tracked in any subsequent experiment.
DNA labeling
(Fig. 8) can involve
the use of radioactive (e.g.,
32
P,
35
S, represented by the black circle in Fig. 8) or
molecular fluorescent labels. Tagging or labeling can be accomplished in one of three
ways. The end labeling technique involves the use of the enzyme, polynucleotide kinase,
which exchanges the unlabeled 5ƍ phosphate group at the end of a target ssDNA or
dsDNA molecule for the terminal
32
P of labeled adenosine triphosphate (ATP), resulting
in
32
P-labeled DNA and unlabeled adeosine diphosphate (ADP) (Fig. 8). The polymerase-
based method utilizes DNA polymerase I Klenow subunit enzyme, which adds
deoxyribonucleotides (A, T, G, C) and creates a complementary strand from a ssDNA
template. If the nucleotides are labeled with either a radioactive or fluorescent label, then
the label will be incorporated into the newly synthesized strand of DNA and the resulting
dsDNA will be labeled on one strand. Finally, the nick translation method utilizes
dsDNA, DNAase I endonuclease, and DNA polymerase I. Here, the DNAase I
endonuclease introduces a limited number of single-stranded breaks (“nicks,” shown as
triangles in Fig. 8) in the DNA molecule, leaving exposed 5ƍ phosphate and 3ƍ hydroxyl
groups. One then adds DNA polymerase I Klenow subunit enzyme, which then adds
labeled nucleotides to the nicked regions and fills in the gap in the DNA strand. Thus,
with any of these methods, we can produce a DNA fragment that we can trace throughout
isolation and manipulation processes via the incorporated label.
Using above-ambient temperatures, we can induce dsDNA in solution to partially
unwind or unfold (“denature”), exposing its single strands. These exposed single strands
can now form base pairs with other DNA or RNA single strands if we first introduce the
“new” DNA or RNA at above ambient temperatures, then slowly lower the temperature
and allow the new + old strands to bind or anneal together during the cooling stage. This
process of annealing or hybridizing DNA and/or RNA strands together is called nucleic
acid hybridization and this technique permits us to identify new pieces of DNA or RNA
which have base pair complementarity to the target DNA or RNA. This method exploits
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