Biomedical Engineering Reference
In-Depth Information
At this time, the deoxynucleotides are added to the growing chain and are
complementary to the nucleotides in the template DNA. The creation of a phospho-
diester bridge between the 3
hydroxyl group at the growing end of the primer and
the 5
phosphate group of the incoming deoxynucleotide elongates the DNA chain,
with the corresponding dNTP releasing pyrophosphate (PPi) when a dNMP is
incorporated (Shendure et al. 2011 ).
The original dideoxy chain termination DNA sequencing technology utilizes
the fact that DNA polymerases will incorporate a chain-terminating
-dideoxynucleotide monophosphate (ddNMP) at the appropriate complemen-
tary position but synthesis will be stopped by the incorporation of the ddNMPs at
the 3
group for dNMP phosphodiester bond formation (Sanger et al. 1977b ; Shendure
et al. 2011 ). Four reactions, containing template, polymerase, all four dNTPs (one
radioactively labeled), and primer, are set up to generate a continuing series of syn-
thesis products that refl ects each potential chain termination position. In addition,
each reaction also contains one of the four ddNTPs at a specifi c ratio refl ecting the
relative probability of incorporation. Many terminated strands of different lengths
exist within each of the four reactions. As each reaction contains only one ddNTP
species, a set of different-length fragments is generated in each reaction, terminated
at all of the positions corresponding to one of the four nucleotides in the template
sequence. The four reactions are then individually separated on a large denaturing
polyacrylamide gel to yield single-nucleotide resolution. The pattern of bands
across the four lanes allows direct readout of the primary sequence of the template
under analysis.
The original radioactive Sanger dideoxy sequencing protocols, which used
end because the next nucleotide to be added lacks the required 3
- 32 P]dATP to label the growing DNA chains, were modifi ed to use [
- 35 S]dATP
or [
emissions of 35 S and 33 P result in
sharper autoradiographic bands, allowing more (longer) sequence to be read
(Shendure et al. 2011 ). Sequences generated using [
- 33 P]dATP because the lower-energy
- 33 P]dATP have short expo-
sure times similar to 32 P, but band resolution comparable to that of 35 S (Zagursky
et al. 1991 ). Another advantage of using 33 P and 35 S is that users are exposed to
lower radiation doses than with 32 P. In lieu of using radioactivity, a chemilumines-
cent detection method was developed that is comparable in sensitivity to traditional
radiolabeling. A biotinylated primer is used in the dideoxy sequencing reactions
and, after electrophoresis of the biotinylated sequencing products on a sequencing
gel, the products are transferred from the gel to a nylon membrane. After UV
cross-linking the DNA to the membrane, the membrane is treated with streptavi-
din, biotinylated alkaline phosphatase, and a detection reagent, such as CDP*
(Tropix/Applied Biosystems), which emits light upon dephosphorylation. After
exposure to X-ray fi lm, the resultant lumigram can be read in a manner similar to
an autoradiogram when using radioactivity (Beck et al. 1989 ; Creasey et al. 1991 ;
Tizard et al. 1990 ). Technology for end labeling of DNA fragments with biotin
allowed this detection method to also be used in chemical sequencing reactions
(Tizard et al. 1990 ).
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