DNA测序

DNA测序（主要内容如下）

·         Sequencing Gel Preparation

·         Preparation of Templates 

·         DNA Sequencing by the Dideoxy Method

·         DNA Sequencing by the Chemical Method

·         Dye Terminator DNA Sequencing

·         Construction of Nested Deletions for Sequencing

·         PCR Sequencing

·         Recipes

·         Q & A Posted in Molecular Biology Method Forum

Sequencing Gel Preparation

·         Sequencing Gel Prep (NWFSC)
Gel preparation, buffer, electrophoresis...

·         Sequencing Gel Prep (NWFSC)
Wedge gel performance without pouring wedge gels

·         Sequencing Gel Prep (Promega)

·         Buffer Gradient Sequencing Gel (Bowtell Lab)

·         Sequencing Gel Prep

·         Buffer Gradient Sequencing Gels (PMCI Research)

Preparation of Templates 

·         Preparation of Template DNA for Sequencing (Promega)

·         PCR Product Sequencing (Genetics Resources CORE Facility at Johns Hopkins U)
Detailed method for preparing PCR products for direct sequencing

·         Plasmid DNA Preparation for Sequencing (Genetics Resources CORE Facility at Johns Hopkins U)

·         Phage and Cosmid DNA Preparation for Sequencing  (Genetics Resources CORE Facility at Johns Hopkins U)

DNA Sequencing by the Dideoxy Method

·         Double Stranded DNA Sequencing (Dideoxy) Double Stranded DNA Sequencing (Dideoxy) ( (Bowtell Lab)

• Sequencing of Double Stranded DNA Using Dideoxy Chain Termination (Donis Keller Lab)
  This method is used to sequence double stranded DNA, such as a plasmid insert or purified PCR product. The dideoxy chain termination (or enzymatic) method of DNA sequencing involves the in vitro synthesis of a DNA strand by a modified bacteriophage T7 DNA polymerase (SequenaseR/USB) using a single stranded DNA template.

·         DNA Sequencing Reaction (Lazo Lab)

·         Old Fashioned Sequencing(Brent Graveley)

·         DNA Sequencing Using an End-Labeled Primer (Promega)

·         DNA Sequencing  Using Direct Incorporation (Promega)

·         Double Stranded DNA Sequencing (Dideoxy) (PMCI Research)

• DNA sequencing (Roe Lab)

                            i.            Bst-catalyzed radiolabeled DNA sequencing

                        ii.            Radiolabeled sequencing gel preparation, loading, and electrophoresis

                    iii.            Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye primers

                        iv.            Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye terminator reactions

                            v.            Sequenase[TM] catalyzed sequencing with dye-labeled terminators

                        vi.            Fluorescent-labeled sequencing gel preparation, pre-electrophoresis, sample loading, electrophoresis, data collection, and analysis on the ABI 373A DNA sequencer

                    vii.            Double-stranded sequencing of cDNA clones containing long poly(A) tails using anchored poly(dT) primers

                viii.            cDNA sequencing based on PCR and random shotgun cloning

DNA Sequencing by the Chemical Method

·         Preparation of G+A Marker
Simplified method for preparing G+A ladder run along with footprinting reaction. It's much simple than the original Maxima-Gilbert sequencing reaction and works fine.

Dye Terminator DNA Sequencing

• Dye Termination Sequencing of dsDNA. (Gimila Lab)
     

·         ABI Dye Primer Reactions using FS (Whitehead Institute/MIT)

·         ABI Dye Primer Reactions using ThermoSequenase(Whitehead Institute/MIT)

·         ABI Dye Primer Reactions Home Brew(Whitehead Institute/MIT)

·         ABI T7 Terminator Reactions(Whitehead Institute/MIT)

·         ABI AmpliTaq Dye Terminator Reactions(Whitehead Institute/MIT)

·         Dye Terminator Cycle Sequencing (PMCI Research)

·         ALF Sequencing with Fluorescein Labelled Nucleotides (PMCI Research)

Construction of Nested Deletions for Sequencing

·         Nested Deletions (Bowtell Lab)
Nested Deletions using exonuclease-III and mung bean nuclease

·         Site-directed Mutagenesis (Promega)
For generation and selection of oligonucleotide-directed mutants

·         Deletion Mutagenesis (Promega)
For construction of plasmid or M13 subclones containing progressive unidirectional deletions of  inserted DNA, Including methods for DNA preparation, vector digestion, exonuclease deletion, ligation,  transformation...

·         Unidirectional deletions (erase-a-base) (Crawford Lab)

PCR Sequencing

·         PEG Purification of PCR Products (GSCWU)
This protocol can be used instead of EXO/SAP for removing excess primers and nucleotides from PCR products before cycle-sequencing

·         Purification and Sequencing of PCR Product (Donis-Keller lab)
This method is used to sequence directly a specific PCR product after large scale amplification. The reaction should be carried out in the usual fashion, except that after optimization of annealing temperature and other conditions in the 5 µl volume, a large scale 50 µl (10X) total reaction volume should be used to generate sufficient PCR product for sequencing.

·         PCR Product Sequencing (Genetics Resources CORE Facility at Johns Hopkins U)
Detailed method for preparing PCR products for direct sequencing

• 96 Well Plate Purification of PCR products
  Purification with Sepahcryl S-300 or S-500 - in filter bottom plates  96-well silent screen plate, Purification with Guanidine-HCl in MES buffer - in glass filter plates
  Purification with Guanidine-HCl in KAc buffer
    

• Preparation of PCR product for direct sequencing (Robert H. Cruickshank)

Chloroform Extraction

Modified Microcon-100 protocol for purification and concentration of DNA from PCR product

Measurement of DNA concentration 

Recipes

• Recipes for Sequencing (Sequencing Group, UW)
  Recipes for gel solutions, loading buffer...