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Primers

PCR primers are important as they are complementary to the beginning and end of the
DNA fragment of interest which one needs to amplify. Primers therefore select the
boundaries of the region to be amplified by PCR. During the PCR annealing cycle, PCR
primers anneal to the complementary region of the DNA. DNA polymerase binding and
the 3 ! of the oligo allows the synthesis of DNA to occur.
PCR Primers are short single"stranded, synthetically synthesi#ed oligonucleotides usually
shorter than $% nucleotides &often '(")$ nucleotides*
Important Factors To Consider When Choosing Primers:
ptimal +m &annealing +* , $%"-%.C
ligonucleotide /C"content should be between 0%"-%1.
Calculated +m &melting temperature* for both primers used in reaction should not
differ 2$.C.
Primer annealing temperature is usually $.C below the calculated lower +m.
!owe3er it should be chosen empirically for indi3idual conditions.
4nner self"complementary hairpins of 20 and of dimers 2( should be a3oided.
35 terminus is e6tremely case sensiti3e " it must not be complementary to any
region of the other primer used in the reaction and must pro3ide correct base
matching to template.
4f possible, primers should start and end with '") / or C residues
Primers should N+ ha3e any e6tensi3e secondary structure or self"
complementarity &can result in Primer"dimers*.
PCR types
Allele-specific PCR7 a diagnostic or cloning techni8ue which is based on single"
nucleotide polymorphisms &9NPs* &single"base differences in DNA*. 4t re8uires
prior :nowledge of a DNA se8uence, including differences between alleles, and
uses primers whose 35 ends encompass the 9NP. PCR amplification under
stringent conditions is much less efficient in the presence of a mismatch between
template and primer, so successful amplification with an 9NP"specific primer
signals presence of the specific 9NP in a se8uence.
Assembly PCR or Polymerase Cycling Assembly (PCA)7 artificial synthesis of
long DNA se8uences by performing PCR on a pool of long oligonucleotides with
short o3erlapping segments. +he oligonucleotides alternate between sense and
antisense directions, and the o3erlapping segments determine the order of the
PCR fragments, thereby selecti3ely producing the final long DNA product.
Asymmetric PCR7 preferentially amplifies one DNA strand in a double"stranded
DNA template. 4t is used in se8uencing and hybridi#ation probing where
amplification of only one of the two complementary strands is re8uired. PCR is
carried out as usual, but with a great e6cess of the primer for the strand targeted
for amplification. ;ecause of the slow &arithmetic* amplification later in the
reaction after the limiting primer has been used up, e6tra cycles of PCR are
re8uired. A recent modification on this process, :nown as Linear"After"The"
E6ponential"PCR &<A+="PCR*, uses a limiting primer with a higher melting
temperature &+m* than the e6cess primer to maintain reaction efficiency as the
limiting primer concentration decreases mid"reaction.
Colony PCR the screening of bacterial &=.Coli* or yeast clones for correct ligation
or plasmid products. 9elected colonies of bacteria or yeast are pic:ed with a
sterile toothpic: or pipette tip from a growth &agarose* plate. +his is then inserted
into the PCR master mi6 or pre"inserted into autocla3ed water. PCR is then
conducted to determine if the colony contains the DNA fragment or plasmid of
interest.
+he Digital polymerase chain reaction simultaneously amplifies thousands of
samples, each in a separate droplet within an emulsion.
Helicase-dependent amplification7 similar to traditional PCR, but uses a constant
temperature rather than cycling through denaturation and annealing>e6tension
cycles. DNA helicase, an en#yme that unwinds DNA, is used in place of thermal
denaturation.
Hot-start PCR7 a techni8ue that reduces non"specific amplification during the
initial set up stages of the PCR. 4t may be performed manually by heating the
reaction components to the melting temperature &e.g., ?$.C* before adding the
polymerase. 9peciali#ed en#yme systems ha3e been de3eloped that inhibit the
polymerase5s acti3ity at ambient temperature, either by the binding of an antibody
or by the presence of co3alently bound inhibitors that only dissociate after a high"
temperature acti3ation step. !ot"start>cold"finish PCR is achie3ed with new
hybrid polymerases that are inacti3e at ambient temperature and are instantly
acti3ated at elongation temperature.
n !it" PCR &49!* is a polymerase chain reaction that actually ta:es place inside
the cell on a slide. n sit" PCR amplification can be performed on fi6ed tissue or
cells.
nterse#"ence-specific PCR &499R*7 a PCR method for DNA fingerprinting that
amplifies regions between simple se8uence repeats to produce a uni8ue
fingerprint of amplified fragment lengths.
n$erse PCR7 is commonly used to identify the flan:ing se8uences around
genomic inserts. 4t in3ol3es a series of DNA digestions and self ligation, resulting
in :nown se8uences at either end of the un:nown se8uence. 4n3erse PCR has
numerous applications in molecular biology including the amplification and
identification of se8uences flan:ing transposable elements, and the identification
of genomic inserts.
+he in3erse PCR method includes a series of digestions and self"ligations with the DNA
being cut by a restriction endonuclease. +his cut results in a :nown se8uence at either end
of un:nown se8uences.
4n3erse PCR 9teps
'* +arget DNA is lightly cut into smaller fragments of se3eral :ilobases by restriction
endonuclease digestion.
)* 9elf"ligation is induced under low concentrations causing the phosphate bac:bone to
reform. +his gi3es a circular DNA ligation product.
3* +arget DNA is then restriction digested with a :nown endonuclease. +his generates a
cut within the :nown internal se8uence generating a linear product with :nown terminal
se8uences. +his can now be used for PCR &polymerase chain reaction*.
0* 9tandard PCR is conducted with primers complementary to the now :nown internal
se8uences.
Ligation-mediated PCR uses small DNA oligonucleotide 5lin:ers5 &or adaptors*
that are first ligated to fragments of the target DNA. PCR primers that anneal to
the lin:er se8uences are then used to amplify the target fragments. +his method is
deployed for DNA se8uencing, genome wal:ing, and DNA footprinting A related
techni8ue is Amplified fragment length polymorphism, which generates diagnostic
fragments of a genome.
%ethylation-specific PCR &%!P* is used to identify patterns of DNA methylation
at cytosine"guanine &Cp/* islands in genomic DNA.+arget DNA is first treated
with sodium bisulfite, which con3erts unmethylated cytosine bases to uracil,
which is complementary to adenosine in PCR primers. +wo amplifications are
then carried out on the bisulfite"treated DNA7 ne primer set anneals to DNA
with cytosines &corresponding to methylated cytosine*, and the other set anneals
to DNA with uracil &corresponding to unmethylated cytosine*. @9P used in A"
PCR pro3ides 8uantitati3e information about the methylation state of a gi3en Cp/
island.
Long PCR is a PCR is which e6tended or longer than standard PCR, meaning
o3er $ :ilobases &fre8uently o3er '% :b*. <ong PCR is usually only useful if it is
accurate. +hus, special mi6tures of proficient polymerases along with accurate
polymerases such as Pfu are often mi6ed together. Applications of Long PCR
<ong PCR is often used to clone larger genes or large segments of DNA which
standard PCR cannot.
%iniprimer PCR7 uses a thermostable polymerase &9"+br* that can e6tend from
short primers &BsmalligosB* as short as ? or '% nucleotides. +his method permits
PCR targeting to smaller primer binding regions, and is used to amplify conser3ed
DNA se8uences, such as the '-9 &or eu:aryotic '(9* rRNA gene.
%"ltiple& Ligation-dependent Probe Amplification &%LPA*7 permits multiple
targets to be amplified with only a single primer pair, thus a3oiding the resolution
limitations of multiple6 PCR &see below*.
%"ltiple&-PCR7 consists of multiple primer sets within a single PCR mi6ture to
produce amplicons of 3arying si#es that are specific to different DNA se8uences.
;y targeting multiple genes at once, additional information may be gained from a
single test run that otherwise would re8uire se3eral times the reagents and more
time to perform. Annealing temperatures for each of the primer sets must be
optimi#ed to wor: correctly within a single reaction, and amplicon si#es, i.e., their
base pair length, should be different enough to form distinct bands when
3isuali#ed by gel electrophoresis.
Nested PCR: increases the specificity of DNA amplification, by reducing
bac:ground due to non"specific amplification of DNA. +wo sets &instead of one
pair* of primers are used in two successi3e PCRs. 4n the first reaction, one pair of
primers is used to generate DNA products, which besides the intended target, may
still consist of non"specifically amplified DNA fragments. +he product&s* are then
used in a second PCR with a set of primers whose binding sites are completely or
partially different from and located 35 of each of the primers used in the first
reaction. Nested PCR is often more successful in specifically amplifying long
DNA fragments than con3entional PCR, but it re8uires more detailed :nowledge
of the target se8uences.
'$erlap-e&tension PCR7 a genetic engineering techni8ue allowing the
construction of a DNA se8uence with an alteration inserted beyond the limit of
the longest practical primer length.
("antitati$e PCR ((-PCR)7 used to measure the 8uantity of a PCR product
&commonly in real"time*. 4t 8uantitati3ely measures starting amounts of DNA,
cDNA or RNA. A"PCR is commonly used to determine whether a DNA se8uence
is present in a sample and the number of its copies in the sample. ("antitati$e
real-time PCR has a 3ery high degree of precision. AR+"PCR methods use
fluorescent dyes, such as 9ybr /reen, =3a/reen or fluorophore"containing DNA
probes, such as +a8@an, to measure the amount of amplified product in real time.
4t is also sometimes abbre3iated to R+"PCR &Real Time PCR* or RA"PCR. AR+"
PCR or R+A"PCR are more appropriate contractions, since R+"PCR commonly
refers to re3erse transcription PCR &see below*, often used in conCunction with A"
PCR.
Re3erse Transcription PCR &R+"PCR*7 for amplifying DNA from RNA. Re3erse
transcriptase re3erse transcribes RNA into cDNA, which is then amplified by
PCR. R+"PCR is widely used in e6pression profiling, to determine the e6pression
of a gene or to identify the se8uence of an RNA transcript, including transcription
start and termination sites. 4f the genomic DNA se8uence of a gene is :nown, R+"
PCR can be used to map the location of e6ons and introns in the gene. +he $5 end
of a gene &corresponding to the transcription start site* is typically identified by
RAC="PCR &Rapid Amplification of cD)A Ends*.
!olid Phase PCR7 encompasses multiple meanings, including Colony
Amplification &where PCR colonies are deri3ed in a gel matri6, for e6ample*,
5;ridge PCR5 &primers are co3alently lin:ed to a solid"support surface*,
con3entional 9olid Phase PCR &where Asymmetric PCR is applied in the presence
of solid support bearing primer with se8uence matching one of the a8ueous
primers* and =nhanced 9olid Phase PCR &where con3entional 9olid Phase PCR
can be impro3ed by employing high +m and nested solid support primer with
optional application of a thermal 5step5 to fa3our solid support priming*.
Thermal asymmetric interlaced PCR (TAL-PCR)7 for isolation of an un:nown
se8uence flan:ing a :nown se8uence. Dithin the :nown se8uence, +A4<"PCR
uses a nested pair of primers with differing annealing temperaturesE a degenerate
primer is used to amplify in the other direction from the un:nown se8uence.
To"chdo*n PCR &!tep-do*n PCR*7 a 3ariant of PCR that aims to reduce
nonspecific bac:ground by gradually lowering the annealing temperature as PCR
cycling progresses. +he annealing temperature at the initial cycles is usually a few
degrees &3"$.C* abo3e the +m of the primers used, while at the later cycles, it is a
few degrees &3"$.C* below the primer +m. +he higher temperatures gi3e greater
specificity for primer binding, and the lower temperatures permit more efficient
amplification from the specific products formed during the initial cycles.
+ni$ersal ,ast -al.ing7 for genome wal:ing and genetic fingerprinting using a
more specific 5two"sided5 PCR than con3entional 5one"sided5 approaches &using
only one gene"specific primer and one general primer " which can lead to
artefactual 5noise5* by 3irtue of a mechanism in3ol3ing lariat structure formation.
9treamlined deri3ati3es of FGD are <aNe RA/= &lariat"dependent nested PCR
for rapid amplification of genomic DNA ends*, $5RAC= <aNe and 35RAC= <aNe.
/ariable )"mber of Tandem Repeats (/)TR) PCR targets areas of the genome
that e6hibit length 3ariation. +he analysis of the genotypes of the sample usually
in3ol3es si#ing of the amplification products by gel electrophoresis. Analysis of
smaller HN+R segments :nown as 9hort +andem Repeats &or 9+Rs* is the basis
for DNA Gingerprinting databases such as CD49.
Pretreatments and extensions
AdCustments to the synthetic oligonucleotides used as primers in PCR are a rich source of
modification7
Normally PCR primers are chosen from an in3ariant part of the genome, and
might be used to amplify a polymorphic area between them. 4n Allele-specific
PCR the opposite is done. At least one of the primers is chosen from a
polymorphic area, with the mutations located at &or near* its 35"end. Fnder
stringent conditions, a mismatched primer will not initiate replication, whereas a
matched primer will. +he appearance of an amplification product therefore
indicates the genotype.
InterSe!ence-Specific PCR &or ISSR-PCR* is method for DNA fingerprinting
that uses primers selected from segments repeated throughout a genome to
produce a uni8ue fingerprint of amplified product lengths. +he use of primers
from a commonly repeated segment is called Al!-PCR, and can help amplify
se8uences adCacent &or between* these repeats.
Primers can also be designed to be 5degenerate5 " able to initiate replication from a
large number of target locations. Whole genome amplification &or W"A* is a
group of procedures that allow amplification to occur at many locations in an
un:nown genome, and which may only be a3ailable in small 8uantities. ther
techni8ues use degenerate primers that are synthesi#ed using multiple
nucleotides at particular positions &the polymerase 5chooses5 the correctly matched
primers*. Also, the primers can be synthesi#ed with the nucleoside analog inosine,
which hybridi#es to three of the four normal bases. A similar techni8ue can force
PCR to perform 9ite"directed mutagenesis.
Normally the primers used in PCR are designed to be fully complementary to the
target. !owe3er, the polymerase is tolerant to mis"matches away from the 35 end.
Tailed-primers include non"complementary se8uences at their $5 ends. A
common procedure is the use of lin#er-primers, which ultimately place
restriction sites at the ends of the PCR products, facilitating their later insertion
into cloning 3ectors.
An e6tension of the 5colony"PCR5 method &abo3e*, is the use of $ector primers.
+arget DNA fragments &or cDNA* are first inserted into a cloning 3ector, and a
single set of primers are designed for the areas of the 3ector flan:ing the insertion
site. Amplification occurs for whate3er DNA has been inserted.
PCR can easily be modified to produce a la%eled prod!ct for subse8uent use as a
hybridi#ation probe. ne or both primers might be used in PCR with a radioacti3e
or fluorescent label already attached, or labels might be added after amplification.
+hese labeling methods can be combined with 5asymmetric"PCR5 &abo3e* to
produce effecti3e hybridi#ation probes.