key: cord-007724-2nwrhk1d
authors: Hofmann, Martin A.; Brian, David A.
title: Sequencing DNA Amplified Directly from a Bacterial Colony
date: 1993
journal: PCR Protocols
DOI: 10.1385/0-89603-244-2:205
sha: 
doc_id: 7724
cord_uid: 2nwrhk1d

A few hundred bacterial cells obtained by touching a bacterial colony with a sterile toothpick can be used directly in a polymerase chain reaction (PCR) amplification procedure to identify and orient a plasmid insert (1,2). By combining this procedure with one in which asymmetrically amplified DNA is used for sequencing (ref. 3 and Fig. 3), we have demonstrated that DNA amplified from a bacterial colony can be sequenced directly by the dideoxy chain-termination method to yield results as good as those obtained when purified template DNA is used for amplification (ref.4 and Fig. 2). By end-labeling the primer that is used in limiting amounts during the amplification step and using it for sequencing, an entire insert of 300 nucleotides or less can be sequenced in one step. Inserts of larger size can be sequenced by using labeled primers that bind within the amplified single-stranded DNA sequence. The procedure is rapid and enables one to obtain sequences from as many as 20 clones in a single day.

A few hundred bacterial cells obtained by touching a bacterial colony with a sterile toothpick can be used directly in a polymerase chain reaction (PCR) amplification procedure to identify and orient a plasmid insert (1, 2) . By combining this procedure with one in which asymmetrically amplified DNA is used for sequencing (ref. 3 and Fig. l) , we have demonstrated that DNA amplified from a bacterial colony can be sequenced directly by the dideoxy chain-termination method to yield results as good as those obtained when purified template DNA is used for amplification (ref. 4 and Fig. 2 ). By end-labeling the primer that is used in limiting amounts during the amplification step and using it for sequencing, an entire insert of 300 nucleotides or less can be sequenced in one step. Inserts of larger size can be sequenced by using labeled primers that bind within the amplified single-stranded DNA sequence. The procedure is rapid and enables one to obtain sequences from as many as 20 clones in a single day. Fig. 1 . The asymmetric PCR reaction. In the procedure described in this chapter, primer 2 is the limiting primer and will be 5' end-lab&d and used in the sequencing reaction for sequencing the single-stranded DNA product.

in water. Approximately 1 mg of oligonucleotide is purified on a NAPS Sephadex G-25 column (Pharmacia, Piscataway, NJ), which has a bed volume of 3 mL. Oligonucleotides are quantitated by spectrophotometry (1 AW U = 33 j.tg/mL) and diluted to a final concentration of 10 p&f or 0.1 pM (depending on the use; see below) in water. Store at -20°C. 1. Prepare a 2OyL end-labeling reaction mix by adding together 1 jtL of 10 pA4 primer 2 (10 pmol), 11 pL of water, 2 pL of 10X kinase buffer, 5 pL of [T-~~P]ATP, 1 pL (10 U) of T4 polynucleotide kinase. This makes enough for 10 sequencing reactions. 2. Incubate at 37°C for 30 min, then add 30 pL of Hz0 to make a total volume of 50 p.L. 3. Purify end-labeled primer by passing it through a BioSpin column that has been equilibrated with water according to manufacturer's instructions. 4. Estimate the volume of eluate and use 0.1 vol (x pL used in Section 3.3., step 1 below) (1 pmol of radiolabeled primer) in the sequencing reaction below. One microliter of the eluate can be counted to determine the specific activity of the radiolabeled primer. Approximately lo6 cpm Cerenkov counts/pmol primer is needed.

This is essentially as described by Innis 

Add 20 pL of PCR mix (from Section 3.1., step 6) containing singlestranded DNA, mix by pipetting 10 times

C reaction tubes, which contain 2.5 pL each of the respective termination mixes and mix by pipetting five times

Terminate the reactions by adding 4 lrL of formamide stop solution. Store at -20°C

For sequencing, the termination mix (10 pL) is heated at 100°C for 3 min and 3 pL/lane is loaded onto a DNA sequencing gel (see Note 2)

pJ4 each) so that residual amounts would not interfere with dideoxynucleotide chain termination reactions (Innis et al., ref. 3). We have learned that when residual amounts do cause a problem (i.e., when short termination products cannot be seen on the sequencing gel), generally as a result of short (~200 nucleotides) inserts in the clone, two approaches can be used to solve this problem: (1) Additional cycles of the PCR (50-60 total) can be run to deplete the dNTPs and (2) The product from the asymmetric reaction

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