Methods and reagents: Cycle sequencing through GC-rich regions
 
Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
Internet.  This month's column discusses a number of methods used to sequence
through GC-rich sections of DNA, and why it is preferable to expose X-ray film
to 32-P at -70 degrees C. For details on how to take part in the newsgroup, see
the accompanying box.

The use of thermal cycling for sequencing DNA fragments with Taq DNA polymerase
by the dideoxynucleotide termination method, also known as cycle sequencing,
has now become standard molecular biology practice.  In many cases, inability
to sequence cloned fragments or PCR-generated double-stranded DNA is hampered
by the inherent nucleotide sequence of the cloned fragment.  If polymerase
stalling occurs during the extension, bands in all four lanes (BAFLs) are seen
on the autoradiograph after the DNA is electrophoresed through a polyacrylamide
gel and the gel is exposed to X-ray film.  The stall point or arrest site on
the X-ray film occurs concomitantly with loss of DNA bands past the stop point
on the gel, obscurring any bands that should have been readable past that
nucleotide position.

GC-rich regions can cause secondary structures, which create problems at
different stages of the polymerase reaction, during (1) the denaturation of the
template, (2) annealing of the primer and (3) extension of the primer by
polymerase.  When encountering troublesome DNAs, netters sometimes use a number
of tricks to read past BAFLs. These include providing a longer denaturation
step and/or performing a hot-start-PCR to keep secondary structures from
forming too quickly, thereby inhibiting the extension (see TiBS 20, 42-44, for
a discussion of hot start PCR).

Additives that increase readability
***********************************
It is well known that adding various substances sometimes helps to increase the
readability of sequence reactions.  For example, co-solvents such as glycerol,
dimethylsulfoxide and formamide work to provide highly stringent conditions for
primer annealing by changing the melting temperature of the primer-template
hybridization reaction.

Co-solvents also have various side effects, such as increasing the
thermostability of the polymerase enzyme, which is really not such a bad
thing.  However, whereas glycerol tends to extend the resistance of Taq
polymerase to heat destruction, formamide lowers enzyme heat-resistance.
In addition, other additives such as spermidine and single-strand binding
protein might help to keep things moving along during the extension by
providing a template free of interfering secondary structures, in the same
manner as an extended denaturation step or hot start would, or perhaps they
free the template from contaminants. [1-3]

One suggestion discussed recently is the use of Perfect Match [R] polymerase
enhancer from Stratagene. This substance is said to decrease mis-primings and,
therefore, spurious banding patterns when added to the PCR. The downside of
using such a kit reagent, however, is that this chemical or enzyme additive, or
whatever it happens to be, is a secret proprietary component and researchers
are provided with little or no information about how it works.

Even though extension reactions can be performed at a high enough temperature
and in combination with additives, which would presumably abrogate any
secondary structure that might cause stalling of the thermostable polymerase,
some unusual DNA structures still can cause premature termination.

Problems with recommended buffers?
**********************************
Genes with either a high GC content or repeated sequences, such as triplet
expansion regions, cause severe problems for sequencing. Recently, it was found
that the recommended buffers commonly used for cycle sequencing might be
responsible for causing stalls within these difficult stretches of DNA.

In one study involving the chicken beta-globin promoter, and in other
unpublished work involving several cloned DNA fragments of the human triplet
expansion region associated with fragile X syndrome, DNA containing GC-rich
stretches always caused difficulty in read-through as a result of strong DNA
synthesis arrest, no matter what polymerase or additive was used.  Use of
Sequitherm [TM], Pfu [TM], Hot Tub [TM], and Vent [TM] polymerases including
gene 32 protein, single-stranded binding protein, 10% dimethylsulfoxide, 10%
glycerol or 10% formamide, had no effect on the arrest. [4]

Interestingly, the arrest site responsible for the stalls was further
determined to be a tetra-plex loop structure, which is stabilized by K+, a
common component used in DNA polymerase buffers.  It was shown that KCl and
potassium glutamate cause stalling, while NaCl, NH4Cl, CsCl, RbCl and SrCl2 do
not.  Use of a K+-free buffer proved successful in obtaining clean results by
cycle sequencing of these genes. [5]

Why expose X-ray film to 32-P at -70 degrees C?
***********************************************
Often people do laboratory techniques out of habit and recently someone
questioned one of many habitual modus operandi - the storage of cassettes at
-70 degrees C when exposing X-ray film to samples radiolabeled with 32-P.
Reflecting, perhaps, how many have simply taken certain techniques as the way
things are done without so much as questioning why, many explanations given by
netters shed very little light on why this is done routinely.

One netter thought that placing the X-ray film in the freezer would improve the
fluorescence of the intensifying screen, but couldn't explain why this is so.
Another person suggested that at -70 degrees C the fluorescent intensifying
screens reflect emitted beta-particles toward the film, effectively emulating a
mirror, and this would cause more energy to be absorbed by the film.  A further
suggestion was that the advantage of doing this was that colder temperatures
would cause beta-particles to have less energy and increase the likelihood of
them interacting with the film and/or the intensifying screen.  Someone else
said that the low temperature improves the efficiency of excitation of the
halides, making the film darker with less exposure time.

Although these answers were thoughtful, many were either partially or entirely
incorrect. In fact, the reasons for exposing X-ray film at -70 degrees C
differs depending on which isotope is used and how much energy it emits.

Converting silver halide crystals
*********************************
When light hits sensitive silver halide grains within the film emulsion, Ag2+
ions are produced as a latent image. These disturbed crystals are converted to
metallic silver much more quickly than undisturbed crystals during development.
[6]

Low energy beta-emitters such as 3-H cannot penetrate through much of the gel
and are thus unable to reach the X-ray film.  Gels with 3-H label are usually
treated with a substance that allows for fluorescent detection.  By contrast,
high energy beta-emitters, such as 32-P, normally pass through the film and are
absorbed by an intensifying screen placed on the other side.  Intensifying
screens, usually composed of calcium tungstate (CaWO4), absorb beta-particles
and emit photons, which create a latent image on the film. The latter is known
as indirect autoradiography, as opposed to direct autoradiography, which is the
conversion of the silver halide crystals by direct hits of beta-particles.

Two photons are usually necessary to convert a single silver halide crystal.
When exposed at -70 degrees C, the half-life of a singly hit Ag2+ ion is longer
than that at room temperature. This allows more time for a second photon to hit
it than at room temperature.  Therefore, while in the freezer, the time-window
for receiving the activation events (photons or beta-particles) is much longer.
[7]

One person mentioned that if the X-ray holder is not fitted with enhancer
screens, there is no reason to place it in the freezer because this will not
make any difference to the image captured on the film.  Others disagreed,
saying that using an intensifying screen is not the only reason to expose at
-70 degrees C.  They said that the film retains an image better at -70 degrees
C, irrespective of whether or not a screen is used. However, when a screen is
not used, only direct autoradiography is possible, which obviates the need for
a longer time-window. [8]

The downside of using the screen is that the latent image created by use of an
enhancing screen (indirect) will be superimposed on the autoradiograph
(direct), and scattering of light will cause loss of resolution to any image.
The trade-off, therefore, is sensitivity versus resolution.

Other drawbacks of putting your cassette in the deep freeze include the time
spent waiting for the cassette to thaw, the risk of fog, smudges and
fingerprints made on the film caused by condensation of water droplets on a
cold piece of film, artifacts created by static electricity discharges, if the
air is dry when opening a cold cassette and the risk of breaking the spine of a
hingeless cassette if it is opened while frozen.

Regardless of why some think this should be done when using 32-P most of the
time, storing the cassettes in the freezer is unnecessary and simply habit.
Most netters feel that this causes more problems than need be. Those who use
quite high-energy radiolabel (more than 50 d.p.m. cm-2) would most likely not
be able to tell the difference between exposures done at -70 degrees C with
screens and those exposed at room temperature without them.

References
**********

[1] Filichkin, S. A. and Gelvin, S. B. (1992)
    BioTechniques 12, 828-830
 
[2] Dutton, C. M., Paynton, C. and Sommer, S. S. (1993)
    Nucleic Acids Res. 21, 2953-2954
 
[3] Wan, C.-Y. and Wilkins, T. A. (1993)
    PCR Methods Appl. 3, 208-210
 
[4] Woodford, K., Howell, R. and Usdin, K. (1994)
    J. Biol. Chem. 269, 27029-27035
 
[5] Woodford, K., Weitzmann, M. N. and Usdin, K. (1995)
    Nucleic Acids Res. 23, 539
 
[6] Jacobson, R. E., Ray, S. F. and Attridge, G. G. (1988)
    in The Manual of Photography, pp. 146, Focal Press
 
[7] Billington, D., Jayson, G. G. and Maltby, P. J. (1992)
    Radioisotopes, pp. 59-60, BIOS Scientific Publishers
 
[8] Laskey, R. A. and Mills, A. D. (1977)
    FEBS Lett. 82, 314-316

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Any statements made by the author are not meant to advocate the use of a
particular commercial product or endorse any company. All opinions are
those of the author and do not reflect the opinion of the National Cancer
Institute or the National Institutes of Health.

Copyright: This manuscript is not copyrighted by Elsevier Publishing Company.
However, you may not reproduce any portion for resale or edit the text for
redistribution, sale, or otherwise without written permission from the 
author.

You found this at the World Wide Web (WWW) Uniform Resource Locator (URL)
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/jan96.txt

Any reference to this column must be cited as the following published article:
Hengen, P. N. 1996. Methods and reagents - Cycle sequencing through GC-rich
regions. Trends in Biochemical Sciences 21(1):33-34.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh@ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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