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Through some quirk of
evolution, DNA polymerase is only able to add nucleotides to the
3' (sugar) end of a growing chain of DNA. Therefore, it is not able
just to
start right up building a complementary strand for a length of DNA that has
been separated from its partner. To get around this problem in
the cell, an enzyme called a primase produces a short
string of nucleotides (usually 15-30) that are complementary to the
first part of the segment of DNA that is being copied. This string of
nucleotides, called a primer ,
attaches to the beginning of the template strand by base pairing. DNA
polymerase is then able to add the next complementary nucleotide to the free 3' end of
the primer. From there it continues adding more complementary nucleotides to the template DNA until a new double strand of
DNA is completed. (See
DNA
Replication for more detail.)
The sides of a DNA molecule are
antiparallel,
that is, they run in
opposite directions. In other words, the sugar molecule of one
base pair compared to the other is flipped over--so that the two
sides of a DNA chain are replicated from different directions.
Since DNA polymerase operates from the 5' (phosphate) to the 3'
(sugar) end, this means that two different DNA primers are
needed--one for each 5' end of side of the molecule.
It is this requirement for dual
primers that makes it possible to precisely define the area of a DNA
molecule to be amplified by the PCR. By using custom made primers with
complementary sequences at, or adjacent to, the beginning of the
marker targeted for amplification, it is possible to specify where
the new chain should begin.
Of course, it is also necessary to
tell the DNA polymerase where to stop as well as where to begin.
A second primer with sequences that are complementary to the end of
the targeted segment is added to the PCR brew. In this way it
is possible to create short segments of "target DNA" that
include only the marker of interest and a bit of its flanking
region on either end. Below is a diagram of the first few
steps of this reaction showing how these primers accomplish this.
The marker in this example is based loosely on the Y STR marker
DYS391 and its published primers
(http://www.cstl.nist.gov/biotech/strbase/str_y391.htm.)
Modifications have been made for brevity and clarity. There
are eight repeats of the tetranucleotide sequence CTAT.
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The marker (DNA
segment) targeted for amplification is highlighted in yellow.
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1 |
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
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Primers to match
the ends of the target are show below--the
left primer is
highlighted in green and the
right primer
highlighted in blue.
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Right and left
arrows signify that the DNA strand extends indefinitely in the
direction of the arrow though sequences are omitted to conserve
space.
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Sequences in green
represent the two strands of the original DNA being amplified.
Sequences in red represent strands synthesized during the PCR
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Each round of the
PCR is a three step process controlled by precise temperature
changes.
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As the temperature
is raised to 94-96° Centigrade, the two strands of DNA separate.
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When the
temperature is lowered to 50-65° Centigrade, the primers find
their complementary sequences adjacent to the target DNA segment
and anneal to them (bind.)
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As the temperature
is raised to 72° Centigrade, DNA polymerase begins adding nucleotides from the PCR
mixture to both strands (see DNA
Replication for more details.)
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12 |
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAA
CTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
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In the first round
(below), there are two double stranded DNA strnds.
However, there is
nothing in this place to tell the enzyme where to stop on either
strand both strands extend well beyond the target segment at the
3' end. However, one end of each newly synthesized strand
has been defined by the positions of the primers--the
leading strand will begin at the 5' end of the target (primer
highlighted in green above.) The lagging strand
(primer highlighted in blue above) will begin at the 3' end. The
original template strands retain their length (signified by the
green arrows.) |
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1
2
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5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
 5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
Results
of Round One
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After round two
(below), two
of the resulting four double stranded molecules each has one strand
that contains only the marker and its flanking regions and one
strand that has one end defined and the other still extended.
The other two strands are each made up of one strand of the
original molecule and one strand that extends beyond the marker at
one end and is "cut" at the other. |
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1
2
3
4
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5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
Results of Round Two
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At the end of round
three (below,) PCR finally yields two double stranded molecules of the
desired length (numbers 3 and 6) and an assortment of others.
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1
2
3
4
5
6
7
8
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5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
5'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-3'
3'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-5'
Results of Round Three |
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As the cycles
continue, more and more of segments that include only the marker
and the bits of flanking nucleotides are generated. While
the original strands do continue to generate "uncut" or
"half-cut" molecules, their concentration is negligible.
At the end of 30 cycles, 1,073,741,764 copies of the desired
length have been produced but only about 60 of the "uncut"
variety.
And, after plowing
through all of this, probably the best way to understand the way
primers can be used to isolate targeted sections of DNA for
amplification in PCR is to view the flash animation from the
Dolan DNA Learning Center at Cold Springs Harbor.
http://www.dnalc.org/shockwave/pcranwhole.html |
It is the sequence of this primer that
determines where it binds to the DNA sample and thereby defines the
region of DNA to be amplified. This small strand of nucleotides
anneals (binds) by complementary base pairing to the beginning of
the area being copied. With the primer in place, DNA
polymerase is then able to continue adding the rest of the pairs of
the segment (Adding an excess of primers insures
that the primers will bind to the separated strands of the DNA
samples so that the separated strands don't just recombine
(self-anneal) when the temperature is lowered.)
By carefully choosing the proper
primers, it is possible to specify exact regions of a DNA mixture
for amplification. So rather than making copies of the entire
mix of DNA in a sample, it is possible to copy only certain segments
which can then be analyzed, measured and compared.
The PCR
process uses primers that have been synthesized in the laboratory to
bind to highly specific sites on DNA. The DNA
between the primers is replicated with great fidelity using a DNA
polymerase enzyme
There are a number of
interactive websites and software programs that aid in designing
primers for replicating specific predefined regions of DNA such as
the markers on the Y chromosome used for genealogical testing.
At present, the application of this science to genealogical research
is very new--with only a handful of labs even offering such
services. It is not clear whether all labs are using the
same primers for each marker. For this reason marker values of
tests conducted by different labs may not be 100% comparable.
For a more
detailed discussion of primer selection see:
http://www.vmresearch.org/genotype/primers.htm
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