Technique / Molecular Biology / DNA analysis techniques / DNA sequencing

DNA sequencing technique methods and troubleshootings

DNA sequencing protocols
(Curr Protoc Mol Biol.)
1: Curr Protoc Mol Biol. 2006 Nov;Chapter 7:Unit 7.8.

Polony DNA sequencing.

Porreca GJ, Shendure J, Church GM.

Harvard Medical School, Boston, Massachusetts, USA.

Polony DNA sequencing provides an inexpensive, accurate, high-throughput way to
resequence genomes of interest by comparison to a reference genome. Mate-paired
in vitro shotgun genomic libraries are produced and clonally amplified on
microbeads by emulsion PCR. These serve as templates for sequencing by
fluorescent nonamer ligation reactions on a microscope slide. Each sequencing run
results in millions of 26-bp reads that can be aligned to the reference genome,
allowing the identification of differences between sequences.


2: Curr Protoc Mol Biol. 2001 May;Chapter 7:Unit7.6.

Denaturing gel electrophoresis for sequencing.

Slatko BE, Albright LM.

New England Biolabs, Beverly, Massachusetts, USA.

The accuracy of DNA sequence determination depends largely upon resolution of the
sequencing products in denaturing polyacrylamide gels. This unit provides a
detailed description of the setup, electrophoresis, and processing of such gels.
In general, the gels required for DNA sequencing are 40-cm long, of uniform
thickness, and contain 4% to 8% acrylamide and 7 M urea. Modifications of this
protocol increase the length of readable sequence information which can be
obtained from a single gel (i.e., forming the gel with wedge-shaped spacers to
create a field gradient, or incorporating a buffer gradient, an electrolyte
gradient, or an acrylamide step gradient into the gel). A modification to the
Basic Protocol--inclusion of formamide in the sequencing gel--is designed to
overcome gel compressions arising from secondary structure in the sequencing
products during gel electrophoresis. A discussion of acrylamide concentrations
and electrophoresis conditions is included in the Commentary.

3: Curr Protoc Mol Biol. 2001 May;Chapter 7:Unit7.5.

DNA sequencing by the chemical method.

Eckert RL.

Case Western Reserve University, Cleveland, Ohio, USA.

This procedure is written for a novice with no chemical sequencing experience. It
is assumed, however, that the investigator has experience with DNA fragment
isolation and DNA labeling with [32P]dNTPs. A DNA fragment labeled at only one
end with 32P or 35S is divided into four aliquots and subjected to base-specific
modification reactions. Piperidine is then added to catalyze strand scission at
the modified bases. Finally, the four reactions are subjected to electrophoresis
on adjacent lanes of a high-resolution denaturing polyacrylamide gel. The gel is
then autoradiographed and the sequence is read from the film.

4: Curr Protoc Mol Biol. 2001 May;Chapter 7:Unit7.4B.

Dideoxy DNA sequencing with chemiluminescent detection.

Martin CS.

Tropix, Inc., Bedford, Massachusetts, USA.

Standard dideoxy DNA sequencing can be performed easily and efficiently with
nonisotopic, chemiluminescent detection by utilizing primers labeled with biotin
in the sequencing reactions. As described in this unit, reaction products are
separated by denaturing gel electrophoresis, transferred to a nylon membrane, and
detected by first binding a streptavidin-alkaline phosphatase conjugate, then
incubating with a chemiluminescent 1,2-dioxetane substrate. The emitted light
signal is imaged on standard X-ray film, producing high-resolution DNA sequencing
ladders. Indirect alkaline phosphatase-labeling of biotinylated DNA with free
streptavidin and biotinylated alkaline phosphatase is also detailed, Finally, the
detection of sequencing reactions labeled with other haptens using specific
antibody-alkaline phosphatase conjugates is described.

5: Curr Protoc Mol Biol. 2001 May;Chapter 7:Unit7.4A.

DNA sequencing by the dideoxy method.

Slatko BE, Albright LM, Tabor S, Ju J.

New England Biolabs, Beverly, Massachusetts, USA.

In the basic dideoxy sequencing reaction, an oligonucleotide primer is annealed
to a single-stranded DNA template and extended by DNA polymerase in the presence
of four deoxyribonucleoside triphosphates (dNTPs), one of which is 35S-labeled.
The reaction also contains one of four dideoxyribonucleoside triphosphates
(ddNTPs), which terminate elongation when incorporated into the growing DNA
chain. After completion of the sequencing reactions, the products are subjected
to electrophoresis on a high-resolution denaturing polyacrylamide gel and then
autoradiographed to visualize the DNA sequence. Three variations of the dideoxy
sequencing procedure are currently in use and are presented in this unit. In the
"labeling/termination" procedure, primer chains are initially extended and
labeled in the absence of terminating ddNTPs, whereas in the traditional "Sanger"
procedure, labeling and termination of primer chains occur in a single step. A
recent variation of the dideoxy sequencing method is thermal cycle sequencing in
which the reaction mixture, containing template DNA, primer, thermostable DNA
polymerase, dNTPs, and ddNTPs, is subjected to repeated rounds of denaturation,
annealing, and elongation steps. The resulting linear amplification of the
sequencing products allows much less template DNA to be used and eliminates
independent primer annealing and template denaturation steps, which are required
for the labeling/termination or Sanger procedures. The use of automated
fluorescent sequencers for four-color dideoxy DNA sequencing is also described in
detail.

6: Curr Protoc Mol Biol. 2001 May;Chapter 7:Unit7.3.

Preparation of templates for DNA sequencing.

Slatko BE, Heinrich P, Nixon BT, Eckert RL.

New England Biolabs, Beverly, Massachusetts, USA.

This unit contains protocols for preparing DNA suitable for use as dideoxy
sequencing templates and as material for end labeling and chemical sequencing. In
all protocols, the starting material contains the recombinant molecule to be
sequenced. DNA from M13mp-derived phage is easily prepared and is currently the
most reliable source of template for large-scale dideoxy sequencing projects.
Because it is occasionally necessary or convenient to use a lambda-derived phage
as a source of DNA, a protocol for preparing lambda phage DNA from plate lysates
is provided. Two protocols for minipreps of plasmid DNA are provided, one
intended for dideoxy sequencing, the other for end labeling and chemical
sequencing; they differ primarily in the way in which cellular RNA is removed.
Alkali denaturation of double-stranded DNA (necessary prior to annealing) is
described, and a final protocol describes the preparation of template for thermal
cycle sequencing from a single phage plaque or bacterial colony.

7: Curr Protoc Mol Biol. 2001 May;Chapter 7:Unit7.2.

Constructing nested deletions for use in DNA sequencing.

Slatko B, Heinrich P, Nixon BT, Voytas D.

New England Biolabs, Beverly, Massachusetts, USA.

Nested deletions useful for dideoxy DNA sequencing are a set of deletions
originating at one end of a target DNA fragment and extending various lengths
along the target DNA. Each successively longer deletion brings "new" regions of
the target DNA into sequencing range (about 300 bp for normal sequencing gels) of
the primer site for a general discussion of nested deletions in DNA sequencing).
Two protocols for generating nested subclones via enzymatic digestion are
included in this unit. In the first, a set of nested deletions is generated by
exonuclease III. The primary advantage of this method is that the deletion
products generated from the original clone can be recircularized to generate
functional plasmids and thus do not require subcloning into another vector. An
alternate method utilizes Bal 31 nuclease to generate the deletions. This method
requires subcloning of the deletion fragments into a separate vector for
subsequent use. Both methods require the presence of unique restriction sites in
the vector that are not present in the insert DNA. Bal 31 can also be used to
generate nested deletions for chemical sequencing in conjunction with specialized
chemical sequencing vectors.

(Selected reviews on DNA sequencing)
1: Metzker ML.
Emerging technologies in DNA sequencing.
Genome Res. 2005 Dec;15(12):1767-76. Review.

2: Tringe SG, Rubin EM.
Metagenomics: DNA sequencing of environmental samples.
Nat Rev Genet. 2005 Nov;6(11):805-14. Review.

3: Edwards JR, Ruparel H, Ju J.
Mass-spectrometry DNA sequencing.
Mutat Res. 2005 Jun 3;573(1-2):3-12. Review.

4: Kan CW, Fredlake CP, Doherty EA, Barron AE.
DNA sequencing and genotyping in miniaturized electrophoresis systems.
Electrophoresis. 2004 Nov;25(21-22):3564-88. Review.

5: Paegel BM, Blazej RG, Mathies RA.
Microfluidic devices for DNA sequencing: sample preparation and electrophoretic
analysis.
Curr Opin Biotechnol. 2003 Feb;14(1):42-50. Review.

6: Franca LT, Carrilho E, Kist TB.
A review of DNA sequencing techniques.
Q Rev Biophys. 2002 May;35(2):169-200. Review.

7: Buchholz BA, Shi W, Barron AE.
Microchannel DNA sequencing matrices with switchable viscosities.
Electrophoresis. 2002 May;23(10):1398-409. Review.

8: Buchholz BA, Barron AE.
The use of light scattering for precise characterization of polymers for DNA
sequencing by capillary electrophoresis.
Electrophoresis. 2001 Nov;22(19):4118-28. Review.

9: Mitnik L, Novotny M, Felten C, Buonocore S, Koutny L, Schmalzing D.
Recent advances in DNA sequencing by capillary and microdevice electrophoresis.
Electrophoresis. 2001 Nov;22(19):4104-17. Review.

10: Marziali A, Akeson M.
New DNA sequencing methods.
Annu Rev Biomed Eng. 2001;3:195-223. Review.

11: Bankier AT.
Shotgun DNA sequencing.
Methods Mol Biol. 2001;167:89-100. Review. No abstract available.

12: Dovichi NJ, Zhang J.
DNA sequencing by capillary array electrophoresis.
Methods Mol Biol. 2001;167:225-39. Review. No abstract available.

13: Watts D, MacBeath JR.
Automated fluorescent DNA sequencing on the ABI PRISM 310 Genetic Analyzer.
Methods Mol Biol. 2001;167:153-70. Review. No abstract available.

14: Messing J.
The universal primers and the shotgun DNA sequencing method.
Methods Mol Biol. 2001;167:13-31. Review. No abstract available.

15: MacBeath JR, Harvey SS, Oldroyd NJ.
Automated fluorescent DNA sequencing on the ABI PRISM 377.
Methods Mol Biol. 2001;167:119-52. Review. No abstract available.

16: Graham CA, Hill AJ.
Introduction to DNA sequencing.
Methods Mol Biol. 2001;167:1-12. Review. No abstract available.

17: Albarghouthi MN, Barron AE.
Polymeric matrices for DNA sequencing by capillary electrophoresis.
Electrophoresis. 2000 Dec;21(18):4096-111. Review.

18: Ronaghi M.
Pyrosequencing sheds light on DNA sequencing.
Genome Res. 2001 Jan;11(1):3-11. Review.

19: Huang GM.
High-throughput DNA sequencing: a genomic data manufacturing process.
DNA Seq. 1999;10(3):149-53. Review.

20: Carrilho E.
DNA sequencing by capillary array electrophoresis and microfabricated array
systems.
Electrophoresis. 2000 Jan;21(1):55-65. Review.

21: Dolnik V.
DNA sequencing by capillary electrophoresis (review).
J Biochem Biophys Methods. 1999 Nov 30;41(2-3):103-19. Review.

22: Schmalzing D, Koutny L, Salas-Solano O, Adourian A, Matsudaira P, Ehrlich
D.
Recent developments in DNA sequencing by capillary and microdevice
electrophoresis.
Electrophoresis. 1999 Oct;20(15-16):3066-77. Review.

DNA sequencing technique books
(Biowww bookshelf)
DNA sequencing application