Methods and reagents: Reamplification of PCR fragments
by Paul N. Hengen, Ph.D. *
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 difficulties encountered by some
netters while trying to reamplify DNA made by the polymerase chain reaction.
For details on how to partake in the newsgroup, see the accompanying box.
Andrew Holyoake
(zool201@csc.canterbury.ac.nz)
was trying to amplify a 1 kb
region of mitochondrial DNA from a single human sperm cell using primers
specific to the target gene. Since the starting template was of such low
quantity and the band of interest was nearly invisible after the primary round
of the polymerase chain reaction (PCR), he was attempting two rounds of
amplification to increase the yield of the desired product.
Highs and woes in PCR-land
As you may have guessed already, a problem occurred when a small portion of the
primary mixture was used as template for reamplification of the desired DNA
fragment - smears were seen on an agarose gel every time. In `Frohmanian'
terms, this common result is often referred to as `ampli-schmutz' [1], and is
mostly caused by nonspecific amplification in the original PCR.
It is also likely that the smears are caused by using far too much DNA in the
secondary PCR. Reamplification works well when the concentration of template
DNA is quite low. Therefore, the primary PCR product must be diluted at least
10,000-fold before reamplification. However, even when the primary mixture of
his DNA was diluted down to as little as 0.1 pg of template DNA, the smear was
not resolved into a discrete band.
Short of suggesting that he should design a new set of oligonucleotide primers
more specific for the target DNA, netters familiar with the problem suggested
that he might improve the yield of PCR product by optimizing the initial
amplification conditions. For example, this may be done by increasing the
annealing temperature, or by decreasing the Mg2+ ion concentration, the
number of cycles, the amount of enzyme, or the concentration of dNTPs. [2]
Initially the Mg2+ concentration was optimized for the primary PCR, although
different concentrations in the secondary PCR also gave a different type of
smear. In addition, changing the annealing temperature, the amount of
polymerase or the number of cycles in either the primary or secondary PCR did
not correct the problem.
Unfortunately, this poor netter suspected that the problem was a combination of
all the various parameters discussed, and he continued to experience the
smears. Someone suggested that he try `touchdown' PCR, a method by which the
annealing temperature is incrementally lowered during each cycle of the PCR,
and that this might help determine the annealing temperature at which a more
specific primary product can be generated. [3,4]
Someone else suggested that chopping up the smear with restriction enzymes
known not to cleave within the PCR product of interest might help alleviate the
smears. Another way would be to design nested primers, which are complementary
to sequences that lie within the fragment generated by the first round of
amplification. [5]
If a DNA band of the expected size can be seen on a gel as well as a background
of smeared DNA, an effective method used to improve reamplification yield and
eliminate most of the unwanted products is gel purification or size
fractionation of the band of interest.
Since slicing a small piece of polyacrylamide gel is very difficult to do, one
person suggested that he extract the DNA from the gel by cutting a trough just
ahead of the migrating DNA band and filling it with low-melting-point agarose.
Electrophoresing the gel horizontally at approximately 10 V cm-1 for 5-10 min
in a submarine-style apparatus would cause the DNA to migrate into the
agarose. The gel slice could then be melted at 65 degrees C and diluted with
Tris-EDTA buffer to recover the DNA. After the amount of DNA has been
estimated, 1-10 ng can be added to a pre-assembled PCR reaction lacking
template for the next round of PCR.
Of course, once the DNA has been electro-eluted from polyacrylamide into
agarose, any of the methods to recover DNA fragments from agarose gels can be
used, as was discussed previously in Methods and reagents [TiBS 19
(1994),388-389]. Alternatively, the remaining DNA from the primary reaction
mixture could be loaded directly onto an agarose gel and the band sliced out.
Unfortunately, a low concentration of PCR product would most probably be
invisible, even with the best detection dye available. If the band could be
seen and extracted, the DNA could be diluted with 1 ml of water and boiled for
5 min before adding 1-5 ul to the other components for the secondary PCR.
An even easier approach is to stick a sterile toothpick or needle into the gel
at the site of the banded fragment, and to swish this through a new PCR
reaction mix in order to inoculate it with a tiny amount of specific DNA. [6,7]
An elegant trick is to have one of the PCR primers biotinylated. 10 ul of
Dynal M-280 streptavidin-coated paramagnetic particles (Dynal, Inc., Great
Neck, NY, USA) are washed in 500 ul of binding buffer (50 mM Tris-HCl, 10 mM
EDTA, 1 M NaCl, pH 7.6). The solution is loaded into a small syringe fitted
with an 18-gauge needle or smaller. A single PCR-amplified band resolved on a
polyacrylamide gel can be recovered by stabbing the needle into the band and
pumping the buffer and beads gently in and out. If the gel is stained with
SYBR Green I (Molecular Probes, Inc., Eugene, OR, USA) and placed on top of a
254 nm transilluminator, the green dye can be seen moving up into the syringe.
Afterwards, the beads are recovered with strong magnets, washed twice with 200
ul of Tris-EDTA, and resuspended in 10 ul of sterile water. Then 1 ul of the
beads is introduced into a fresh PCR mix to reamplify the fragment. After PCR,
the beads can be recovered by magnets or centrifugation and the template can be
reused. In addition, the attached DNA template can be used for sequencing
directly by solid-phase cycle sequencing. [8]
Although Mr Holyoake did not obtain the desired result, he was encouraged by a more positive outcome - another netter experiencing the same problem found a solution. After having difficulty pinpointing a single cause for the smears, careful consideration of all the materials used allowed this netter to determine that one of the contributing factors was an excess of one primer, which may have interfered with the amplification.
References
[1] Frohman, M. A. (1994) in The Polymerase Chain Reaction (Mullis, K. B., Ferre', F., and Gibbs, R. A., eds) p. 14-37, Birkhauser
[2] Carbonari, M. et al. (1993) Trends Genet. 9, 42-43
[3] Don, R. H. et al. (1991) Nucleic Acids Res. 19, 4008
[4] Roux, K. H. (1994) BioTechniques 16, 812-814
[5] Danziger, R. S., Hogarth, L. A., and Star, R. A. (1993) BioTechniques 14, 370
[6] Kadokami, Y. and Lewis, R. V. (1994) BioTechniques 17, 438
[7] Bjourson, A. J. and Cooper, J. E. (1992) Nucleic Acids Res. 20, 4675
[8] Hultman, T. et al. (1989) Nucleic Acids Res. 17, 4937-4946
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.
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You found this at the World Wide Web (WWW) Uniform Resource Locator (URL):
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/mar95.txt
Any reference to this column must be cited as the following published
article:
@article{Hengen1995Martibs,
author = "P. N. Hengen",
title = "Methods and reagents - Reamplification of {PCR} fragments",
journal = "Trends in Biochemical Sciences",
volume = "20",
number = "3",
pages = "124-125",
month = "March",
year = "1995"}
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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 /--------------------------/* * - - - Methods FAQ list -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - - * * - TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - - * * - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/ * ******************************************************************************* |
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