PCR Protocol--Variants of PCR

Variants of PCR

From Wikipedia, the free encyclopedia

This page assumes familiarity with the terms and components used in the Polymerase Chain Reaction (PCR) process.
The versatility of PCR has led to a large number of variants:

Contents
Basic modifications
Pretreatments and extensions
Buffer and temperature modifications
Primer modifications
Polymerase modifications
Mechanism modifications
Isothermal amplification methods
Additional reading
References
Basic modifications
Often only a small modification needs to be made to the 'standard' PCR protocol to achieve a desired goal:
One of the first adjustments made to PCR was the amplification of more than one target in a single tube. Multiplex-PCR can involve up to a dozen pairs of primers acting independently. This modification might be used simply to avoid having to prepare many individual reactions, or could allow the simultaneous analysis of multiple targets in a sample that has only a single copy of a genome. In testing for genetic disease mutations, six or more amplifications might be combined. In the standard protocol for DNA Fingerprinting, the 13 targets assayed are often amplified in groups of 3 or 4. Multiplex Ligation-dependent Probe Amplification (or MLPA) permits multiple targets to be amplified using only a single pair or primers, avoiding the resolution limitations of multiplex PCR.
VNTR PCR involves few modifications to the basic PCR process, but instead targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves simple sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as Short Tandem Repeats (or STRs) is the basis for DNA Fingerprinting databases such as CODIS.
Asymmetric PCR is used to preferentially amplify one strand of the target DNA. It finds use in some types of sequencing and hybridization probing, where having only one of the two complementary strands of the product is advantageous. PCR is carried out as usual, but with a limiting amount of one of the primers. When it becomes depleted, continued replication leads to an arithmetic increase in extension of the other primer[1]. A recent modification on this process, known as Linear-After-The-Exponential-PCR (or LATE-PCR), uses a limiting primer with a higher melting temperature Melting temperature (or Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction[2]. (Also see Overlap-extension PCR).
Some modifications are needed to perform long PCR. The original Klenow-based PCR process had trouble making a product larger than about 400 bp. However, early characterization of Taq polymerase showed that it could amplify targets up to several thousand bp long[3]. Since then, modified protocols have allowed targets of over 50,000 bp to be amplified[4].


Nested PCR
Nested PCR, another early modification, can be used to increase the specificity of DNA amplification. Two sets of primers are used in two successive reactions. In the first, one pair of primers is used to generate DNA products, which may also contain products amplified from non-target areas. The products from the first PCR are then used to start a second, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set. The specificity of all of the primers is combined, usually leading to a single product. Nested PCR is often more successful in specifically amplifying long DNA products than conventional PCR, but it requires more detailed knowledge of the sequence of the target.
Quantitative PCR (or Q-PCR) is used to measure the specific amount of target DNA (or RNA) in a sample. The normal PCR process is performed in a way that is largely qualitative - the amount of final product is only slightly proportional to the initial amount of target. By carefully running the amplification only within the phase of true exponential increase (avoiding the later 'plateau' phase), the amount of product is more proportional to the initial amount of target. Thermal cyclers have been developed which can monitor the amount of product during the amplification, allowing quantitation of samples containing a wide range of target copies. A method currently used is Quantitative Real-Time PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product as the amplification progresses. It is often confusingly referred to as RT-PCR, the same acronym used for PCR combined with Reverse Transcriptase (see below), which itself might be used in conjunction with Q-PCR. More appropriate acronyms are QRT-PCR or RTQ-PCR.
Hot-start PCR is a technique that modifies the way that a PCR mixture is initially heated. During this step the polymerase is active, but the target has not yet been denatured and the primers may be able to bind to non-specific locations (or even to each other). The technique can be performed manually by heating the reaction components to the melting temperature (e.g. 95°C) before adding the polymerase[5]. Alternatively, specialized systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody, or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. 'Hot-start/cold-finish PCR' is achieved with new hybrid polymerases that are inactive at ambient temperature and are only activated at elevated temperatures.
Another simple modification can also decrease non-specific amplification. In Touchdown PCR, the temperature used to anneal the primers is gradually decreased in later cycles. The annealing temperature in the early cycles is usually 3-5°C above the standard Tm of the primers used, while in the later cycles it is a similar amount below the Tm. The initial higher annealing temperature leads to greater specificity for primer binding, while the lower temperatures permit more efficient amplification to the end of the reaction[6].
Other common modifications to PCR allow it to amplify low copy targets. The original report on Taq polymerase[3] showed how the use of up to 60 cycles could amplify targets diluted to just one copy per reaction tube. A later report[7] showed how multiple genetic loci could be amplified and analyzed from a single sperm. Modified protocols[8] have allowed the identification of just one copy of the HIV genome within the DNA of up to 70,000 host cells.
Assembly PCR (also known as Polymerase Cycling Assembly or PCA) is the artificial synthesis of long DNA structures by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotide building blocks alternate between sense and antisense directions, and the overlaps determine the order of oligonucleotides, thereby selectively producing the final long DNA product[9].
In Colony PCR, bacterial colonies are rapidly screened by PCR for correct DNA vector constructs. Colonies are sampled with a sterile toothpick and dabbed into a master mix. To free the DNA for amplification, PCR is either started with an extended time at 95°C (when standard polymerase is used), or with a shortened denaturation step at 100°C and special chimeric DNA polymerase[10]. Colonies from the master mix that shows the desired product are then tested individually.
The Digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion.
Pretreatments and extensions
The basic PCR process can sometimes precede or follow another technique:
RT-PCR (or Reverse Transcription PCR) is a common method used to amplify, isolate, or identify a known sequence from a cell's or tissue's RNA. PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into cDNA. The two reactions are compatible enough that they can be run in the same mixture tube, with the initial heating step of PCR being used to inactivate the transcriptase[3]. Also, the Tth polymerase described below exhibits RT activity, and can carry out the entire combined reaction. RT-PCR is widely used in expression profiling, which determines the expression of a gene or identifies the sequence of an RNA transcript (including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene). The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method named RACE-PCR, short for Rapid Amplification of cDNA Ends. (Note that the acronym RT-PCR has more recently been applied to Real-Time PCR, a version of Quantitative PCR described above.)
Since PCR is based on components of DNA replication, it is not surprising that it can easily be combined[1] with DNA sequencing. In its simplest form, the products of an 'asymmetric PCR' (above) are diluted into a new reaction containing sequencing components, which are then extended by Taq polymerase.
Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' that are first ligated to fragments of the target DNA. PCR primers are then chosen from the linker sequences, and used to amplify the unknown target fragments. It has been used for DNA sequencing, genome walking, and DNA footprinting[11]. A related technique, Amplified fragment length polymorphism, looks at fragments of a genome that differ in length.
Methylation-specific PCR (or MSP) was developed to study patterns of methylation at CpG islands in genomic DNA[12]. Target DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two amplifications are then carried out on the modified DNA, using primer sets that distinguish between the modified and unmodified templates. One primer set recognizes DNA with cytosines to amplify the previously methylated DNA, and the other set recognizes DNA with uracil or thymine to amplify unmethylated target. MSP using Q-PCR can also be performed to obtain quantitative information about methylation.
Buffer and temperature modifications
Adjustments to the 'small' components in PCR can sometimes be useful:
The divalent magnesium ion (Mg++) is crucial to the activity of the polymerase used in PCR. Since many of the other components used in an amplification will also bind Mg++, it's exact concentration available to the enzyme is difficult to control. In general, lower concentrations will increase replication fidelity, while higher concentrations will introduce more mutations (either of which may be desired).
The use in PCR of modified dNTPs can help to control 'carryover' contamination. The PCR process can be carried out using dUTP, an analog of the normal dTTP. Later amplifications are then treated with an enzyme that destroys DNA containing the analog, but leaving the normal target DNA unmodified[13]. Thus, targets that represent contamination from earlier amplifications are selectively destroyed.
A wide variety of other chemicals can be added to PCR, for a variety of effects. Mild denaturants (such as DMSO) can increase amplification specificity by destabilizing non-specific primer binding. Certain chemicals (such as glycerol) can act as stabilizers for the activity of the polymerase during amplification. Detergents (such as Triton X-100) can prevent having the polymerase stick to itself, or to the walls of the reaction tube.
The temperature changes carried out by the thermal cycler will also affect amplification. A particular set of primers are usually tested using different annealing temperatures to determine their optimum. The time given to the polymerase to fully copy the templates may need to be adjusted, depending on their lengths. Longer extension times can also lead to higher yields after the reaction has entered the 'plateau' phase. When amplifying low-copy targets, the total number of cycles performed must be increased.
The polymerases that perform replication during PCR sometime incorporate incorrect bases. This is of no consequence to most assays that test the bulk of the amplified product - the errors are scattered within the product at random, and aren't seen by the assay. However, it is best to perform high-fidelity PCR when the products are individually cloned (for sequencing or expression). A different DNA polymerase (such as Pfu, with a proofreading activity missing in Taq) might be used, and the Mg++ and dNTP concentrations might be adjusted to maximize the number of products that exactly match the original target DNA. Some researchers choose to do the opposite, purposefully running PCR under low-fidelity conditions to produce a spectrum of mutations in the amplified product.
(For additional details, see the auxiliary article PCR optimization.)
Primer modifications
Adjustments to the synthetic oligonucleotides used as primers in PCR are a rich source of modification:
Normally PCR primers are chosen from an invariant part of the genome, and might be used to amplify a polymorphic area between them. In 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 3'-end. Under stringent conditions, a mismatched primer will not initiate replication, whereas a matched primer will. The appearance of an amplification product therefore indicates the genotype. (For more information, see SNP genotyping.)
InterSequence-Specific PCR (or ISSR-PCR) is method for DNA fingerprinting that uses primers selected from segments repeated throughout a genome to produce a unique fingerprint of amplified product lengths[14]. The use of primers from a commonly repeated segment is called Alu-PCR, and can help amplify sequences adjacent (or between) these repeats.
Primers can also be designed to be 'degenerate' - able to initiate replication from a large number of target locations. Whole genome amplification (or WGA) is a group of procedures that allow amplification to occur at many locations in an unknown genome, and which may only be available in small quantities. Other techniques use degenerate primers that are synthesized using multiple nucleotides at particular positions (the polymerase 'chooses' the correctly matched primers). Also, the primers can be synthesized with the nucleoside analog inosine, which hybridizes to three of the four normal bases. A similar technique can force PCR to perform Site-directed mutagenesis. (also see Overlap extension polymerase chain reaction)
Normally the primers used in PCR are designed to be fully complementary to the target. However, the polymerase is tolerant to mis-matches away from the 3' end. Tailed-primers include non-complementary sequences at their 5' ends. A common procedure is the use of linker-primers, which ultimately place restriction sites at the ends of the PCR products, facilitating their later insertion into cloning vectors.
An extension of the 'colony-PCR' method (above), is the use of vector primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector, and a single set of primers are designed for the areas of the vector flanking the insertion site. Amplification occurs for whatever DNA has been inserted[3].
PCR can easily be modified to produce a labeled product for subsequent use as a hybridization probe. One or both primers might be used in PCR with a radioactive or fluorescent label already attached, or labels might be added after amplification. These labeling methods can be combined with 'asymmetric-PCR' (above) to produce effective hybridization probes.
Polymerase modifications
There are many choices for the all-important DNA polymerase used in PCR:
The Klenow fragment, derived from the original DNA Polymerase I from E. coli, was the first enzyme used to demonstrate PCR. It is inactivated in the denaturation step of PCR, and had to be replenished during each cycle.
The bacteriophage T4 DNA polymerase was also tested shortly after the first reports of PCR. It has a higher fidelity of replication than the Klenow fragment. Since it is also destroyed by heat, it has seen little use since the development of thermostable polymerases.
The DNA polymerase from Thermus aquaticus (or Taq), was the first thermostable polymerase used in PCR[3], and is still the one most commonly used. The enzyme can be isolated from its 'native' bacterial source, or from a cloned gene expressed in E. coli.
The Stoffel fragment is produced from a truncated gene for Taq polymerase, expressed in E. coli. It is missing the 'forward' nuclease activity, and may be able to amplify longer targets than the native enzyme.
The Faststart polymerase is a variant of Taq polymerase that only becomes active after the first denaturation step of PCR, thereby avoiding problems during the first cycle. (see Hot-start PCR above)
A thermostable polymerase has also been isolated from the archeozoic organism Pyrococcus furiosus. Unlike Taq polymerase, Pfu DNA polymerase includes a 'proofreading' activity, leading to about a 5-fold decrease in the error rate of replication[15]. Since these errors accumulate during every cycle of PCR, Pfu is the preferred polymerase when products are to be individually cloned for sequencing or expression.
An extremely thermostable DNA polymerase has been isolated from Thermococcus litoralis, and is marketed as Vent polymerase.
Another thermostable polymerase has been isolated from Thermus thermophilus, and is known as Tth polymerase. In the presence of Mn++ ions, it exhibits a reverse transcriptase activity, allowing PCR amplification to be initiated by RNA targets.
But not Bst polymerase, isolated from the thermophilic bacterium Bacillus stearothermophilus. This was an early candidate to be tested for PCR. It was later found to be unsuitable for continued amplification - it is irreversibly inactivated during the denaturation step. This highlights the point that a good polymerase for PCR should both be active at a higher temperature (for specificity), and should also be able to survive the near-boiling temperatures of the PCR process.
Mechanism modifications
Sometimes even the basic mechanism of PCR can be modified:
Unlike normal PCR, Inverse PCR allows amplification and sequencing of DNA that surrounds a known sequence. It involves initially subjecting the target DNA to a series of restriction enzyme digestions, and then circularizing the resulting fragments by self ligation. Primers are designed to be extended outward from the known segment, resulting in amplification of the rest of the circle. This is especially useful in identifying sequences to either side of various genomic inserts[16].
Similarly, Thermal Asymmetric InterLaced PCR (or TAIL-PCR) is used to isolate unknown sequences flanking a known area of the genome. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures. A 'degenerate' primer is used to amplify in the other direction from the unknown sequence[17].
Isothermal amplification methods
Some amplification protocols have been developed that only remotely resemble PCR:
Helicase-dependent amplification is a technique that is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension steps. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation[18].
PAN-AC also uses isothermal conditions for amplification, and may be used to analyze living cells[19][20].
Additional reading
PCR Applications Manual (from Roche Diagnostics).]

[edit] References
1. ^ a b Innis MA, Myambo KB, Gelfand DH, Brow MA. (1988). "DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA". Proc Natl Acad Sci USA 85: 9436-4940. PMID 3200828.
2. ^ Pierce KE and Wangh LJ (2007). "Linear-after-the-exponential polymerase chain reaction and allied technologies Real-time detection strategies for rapid, reliable diagnosis from single cells". Methods Mol Med. 132: 65-85. PMID 17876077.
3. ^ a b c d e Saiki et al. "Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase." Science vol. 239 pp. 487-91 (1988).
4. ^ Cheng S, Fockler C, Barnes WM, Higuchi R. "Effective amplification of long targets from cloned inserts and human genomic DNA." Proc Natl Acad Sci vol. 91(12) pp. 5695-9 (1994).
5. ^ Q. Chou, M. Russell, D.E. Birch, J. Raymond and W. Bloch (1992). "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications". Nucleic Acids Research 20: 1717-1723.
6. ^ Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS (1991). "'Touchdown' PCR to circumvent spurious priming during gene amplification.". Nucl Acids Res 19: 4008.
7. ^ Boehnke M et al. "Fine-structure genetic mapping of human chromosomes using the polymerase chain reaction on single sperm." Am J Hum Genet vol. 45(1) pp. 21-32 (1989).
8. ^ Kwok S et al. "Identification of HIV sequences by using in vitro enzymatic amplification and oligomer cleavage detection." J. Virol. vol. 61(5) pp. 1690-4 (1987).
9. ^ Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995). "Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides". Gene 164: 49-53. PMID 7590320.
10. ^ Pavlov AR, Pavlova NV, Kozyavkin SA, Slesarev AI (2006). "Thermostable DNA Polymerases for a Wide Spectrum of Applications: Comparison of a Robust Hybrid TopoTaq to other enzymes", in Kieleczawa J: DNA Sequencing II: Optimizing Preparation and Cleanup. Jones and Bartlett, pp. 241-257. ISBN 0-7637338-3-0.
11. ^ Mueller PR, Wold B (1988). "In vivo footprinting of a muscle specific enhancer by ligation mediated PCR". Science 246: 780-786. PMID 2814500.
12. ^ Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB (1996). "Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands". Proc Natl Acad Sci U S A 93 (13): 9821-9826. PMID 8790415.
13. ^ Longo MC, Berninger MS, Hartley JL "Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions." Gene vol. 93(1) pp. 125-8 (1990).
14. ^ E. Zietkiewicz, A. Rafalski, and D. Labuda (1994). "Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification". Genomics 20 (2): 176-83.
15. ^ Cline J,Braman JC, Hogrefe HH "PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases." Nucleic Acids Research vol. 24(18) pp. 3546-51 (1996).
16. ^ Ochman H, Gerber AS, Hartl DL (1988). "Genetic applications of an inverse polymerase chain reaction". Genetics 120: 621-623. PMID 2852134.
17. ^ Y.G. Liu and R. F. Whittier (1995). "Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking". Genomics 25 (3): 674-81.
18. ^ Myriam Vincent, Yan Xu and Huimin Kong (2004). "Helicase-dependent isothermal DNA amplification". EMBO reports 5 (8): 795–800.
19. ^ David, F.Turlotte, E., (1998). "An Isothermal Amplification Method". C.R.Acad. Sci Paris, Life Science 321 (1): 909-914.
20. ^ Fabrice David (September-October 2002). Utiliser les propriétés topologiques de l’ADN: une nouvelle arme contre les agents pathogènes. Fusion.(in French)
Retrieved from "http://en.wikipedia.org/wiki/Variants_of_PCR"

Introduction o PCR

General PCR introduction

1. IntroductionIn 1983 Kary B. Mullis was driving through California on a moonlight night (Mullis, 1990). He was pondering how to use DNA polymerase with oligonucleotide primers in order to identify a given nucleotide at a given position in a complex DNA molecule, such as the human genome. During this drive he invented or discovered the elegant method of making unlimited DNA copies from a single copy of DNA, and called the method: "Polymerase Chain Reaction" (PCR). A couple of months later he conducted the first successful experiment. Ten years after his drive in California, he was awarded the Nobel Prize in Stockholm for his brilliant discovery (Carr, 1993).
PCR was first published in 1985 (Saiki et al., 1985) with Klenow polymerase used as the elongation enzyme. Due to the heat instability of the Klenow polymerase, new enzyme had to be added for every new cycle, and the maximum limit of the product length was 400 bp. In 1988 the first report using DNA polymerase from Thermophilus aquaticus (Taq-polymerase) was published (Saiki et al., 1988). This polymerase greatly enhanced the value of PCR, and the introduction of the automatic programmable heating block in the same report also took the tedious need for three different water baths out of the procedure. Currently the PCR technique is utilized in most molecular biology laboratories as a routine tool which is suitable for performing a great number of different experiments. The method is frequently chosen for conducting experiments, such as cloning, making mutations, sequencing, detecting, typing, etc. (Erlich et al., 1991).

2. AnimationThe basic molecular events of PCR are illustrated in an animation of the liquid phase DNA amplification, which is a prerequisite of the solid phase DNA amplification. The whole animation can be seen in the DIAPOPS animation.

3. The basic reactionPCR is based on the recognition by a short piece of DNA (the primer) of a sequence on a larger, single stranded fragment of DNA (template strand). When the primer recognizes the template and binds (anneals) to the recognition sequence, the 3'-end of the primer is used by DNA polymerase to synthesize a new DNA strand (elongation). When the temperature is raised, the new DNA strand will melt away (denature) from the template, and the template is once again open for annealing of a new primer when the temperature is decreased. By adding a second primer which recognizes the template strand complementary to the first template, the elongation can proceed in the direction of the first primer. In the first round of elongation, this will ideally double the amount of template strands. In the second temperature cycling, half of the templates for the first primer will be new-synthesized fragments, all terminated where the second primer annealed. When these new fragments are recognized by the first primer, the elongation cannot proceed beyond the second primer, and the synthesized fragments will have a fixed length determined by the distance of the annealing sites of the two primers. New production of template strands take place in every temperature cycle. In this way the DNA sequence between the two primer sequences is amplified exponentially, yielding high concentrations of double-stranded DNA of the same length. The newly-formed double stranded DNA is denatured at 94-97ºC. Primers anneal at 35-72ºC (the exact temperature is primer- and assay dependent), and the new product is synthesized at 72ºC, which is the optimal temperature for the Taq-polymerase.

4. ConclusionPCR is capable of producing large amounts of DNA fragments from a single piece of template DNA as the amplification increases the amount of fragments produced exponentially. In theory, it is possible to detect a single copy of template DNA by PCR using simple methods. For this reason PCR is used to identify nucleic acid sequences that are only present in very small numbers in the sample to be analyzed.

Lecture of PCR-2
Introduction to PCR. Molecular biology relies on techniques that enable the detection or ... With the introduction of the Polymerase Chain Reaction (PCR), ...www.modares.ac.ir/elearning/mnaderi/Genetic%20Engineering%20course%20II/Pages/Lecture2.htm
PCR Technology
Introduction. Polymerase chain reaction (PCR) has rapidly become one of the most widely used techniques in molecular biology and for good reason: it is a ...www.accessexcellence.org/LC/SS/PS/PCR/PCR_technology.html
Introduction to PCR
Either way, the DNA is extracted from the source and is amplified via PCR (the Polymerase Chain Reaction). This allows very minute amounts of DNA to be ...nature.umesci.maine.edu/forensics/p_intro.htm
6.1 Polymerase Chain Reaction (PCR) Introduction6.1 Polymerase Chain Reaction (PCR). Introduction. T. he polymerase chain reaction technique employs oligonucleotide primers to amplify segments of ...www.fws.gov/policy/library/fh_handbook/Volume_1/Chapter_6.pdf
Real-Time PCR Introduction [M.Tevfik DORAK]
Overview by MT Dorak, University of Alabama at Birmingham, USA.dorakmt.tripod.com/genetics/realtime.html
YouTube - EDIROL PCR Introduction
This is a video introduction to our new PCR MIDI controllers.www.youtube.com/watch?v=vfiK7Fl75ZQ

PCR Protocol--PCR SSCP

PCR SSCP

Protocol: Mutation Detection by SSCP PCR.
A protocol for mutation detection by single-strand conformational polymorphism (SSCP) by PCR from the neurogenetics laboratory in the Neurological Sciences ...www.ohsu.edu/nsi/faculty/reddyh/lab/protsscp.html
step by step sscp
Step by Step SSCP. Travis Glenn. Laboratory of Molecular Systematics. Smithsonian Institution. Washington, DC 20560. phone: 301-238-3444. fax: 301-238-3059 ...www.uga.edu/srel/DNA_Lab/SSCP'96V2.rtf
Springer Protocols: Abstract: Multiple Fluorescence-Based PCR-SSCP analysis with primer, post- and internal labeling.
Springer Protocols is the largest subscription-based electronic database of reproducible laboratory protocols in the Life and Biomedical Sciences.www.springerprotocols.com/Abstract/doi/10.1385/0-89603-499-2:51
PCR-SSCP: a practical approach (detailed SSCP protocols)
The multiplexed PCR-SSCP analysis described here (Protocol 5) is essentially a two step procedure, each strand of the target DNA sequence is labelled as it ...europium.csc.mrc.ac.uk/WebPages/Database/Methods/pcrpract.htm
Optimization of Nonisotopic PCR–Single-Strand Conformation Polymorphism.
The protocol used for the GCK gene allowed us to establish a successful strategy for the development of PCR-SSCP on other genes such as BRCA1 (breast cancer ...www.clinchem.org/cgi/content/full/43/11/2190
Genomic Variation Laboratory – SSCP Protocol
Feb 27, 2003 ... SSCP Protocol. MDE gel (BioWhittaker Molecular Applications) final ... 3) For single strands, mix 2 ul PCR product for each sample with 10 ...http://genome-lab.ucdavis.edu/Protocols/SSCP%20Protocols.htm
Evaluating Duplicate Gene Expression using RT-PCR/SSCP Analysis (Wendel Lab.)
The subsequent PCR reaction follows the protocol you have predetermined to work best .... 1995, Identification of DNA polymorphism by asymmetric-PCR SSCP. ...
http://www.eeob.iastate.edu/faculty/WendelJ/rt-pcr_sscp.htm
Sensitive detection of p53 gene mutations by a 'mutant enriched PCR SSCP technique.
In the past, the existing PCR-SSCP technique as established by Orita et al. ... Figure 1 gives a schematic view of the protocol applied whereby the ...nar.oxfordjournals.org/cgi/content/full/26/5/1356
Single-strand conformational polymorphism.
cantly to its utility. SSCP PROTOCOL. The following is an example of an SSCP protocol that we used for detection. $138 PCR Methods and Applications ...www.genome.org/cgi/reprint/4/3/S137.pdf?ck=nck

PCR Protocol--PCR RFLP

PCR RFLP

Restriction fragment length polymorphism (From Wikipedia, the free encyclopedia):
A Restriction Fragment Length Polymorphism (or RFLP, often pronounced as "rif-lip") is a variation in the DNA sequence of a genome which can be detected by a laboratory technique known as gel electrophoresis. Analysis of RFLP variation was an important tool in genome mapping, localization of genetic disease genes, determination of risk for a disease, genetic fingerprinting, and paternity testing.
Contents
1 Analysis technique
2 Examples
3 Applications
4 Alternatives
5 References
5.1 External links

PCR-RFLP Method
18-Oct-02.www.ihwg.org/components/cytokine/mon8Lin.htm
Examples of PCR-RFLP for Nematode Diagnostics
Examples of Restriction Fragment Length Polymorphism (RFLP)electrophoresis slabs for different nematodes, from University of Nebrasca.nematode.unl.edu/its_id/EXAMPLES/index.htm
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences
Detection of Point Mutations by RFLP of PCR Amplified DNA Sequences.www.kfunigraz.ac.at/~binder/thesis/node64.html
RFLP/PCR Polymorphism Query Form
Search for RFLP and PCR based polymorphisms by strain, locus symbol, or map position. To search for SNPS, use the SNP Query Form. ...www.informatics.jax.org/searches/polymorphism_form.shtml
PCR, RFLP and Gene Therapy
Lecture 24:Genetic Engineering: PCR, RFLP Analysis & Gene Therapy. The Polymerase Chain Reaction (PCR) Can Make Millions of Copies of DNA in a Short Time ...http://members.aol.com/BearFlag45/Biology1A/LectureNotes/lec24.html
Handbook for DNA isolation, RAPD-PCR and PCR-RFLP
General protocol. We use agarose gels for checking the quality of DNA isolates, PCR products, and PCR-RFLP products, and for scoring RAPD products. ...www.toyen.uio.no/botanisk/brochmann/handbook.htm
PCR-RFLP PROTOCOL FOR ALLELES A AND B PCR ...BOVINE KAPPA-CASEIN PCR-RFLP PROTOCOL. FOR ALLELES A AND B. Laboratory of J.F. Medrano. Department of Animal Science. University of California. ...animalscience.ucdavis.edu/laboratory/

PCR Protocol--Competitive Quantitative RT-PCR

Competitive and/or Quantitative RT-PCR

Competitive RT-PCR (Dieter Kaufmann Lab.)

Quantitative RT-PCR (Morimoto Lab.)

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Other PCR Procedures (Jack Vanden Heuvel Lab.)
http://www.cas.psu.edu/docs/CASDEPT/VET/jackvh/jvhpcr.html

Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample (Biological Procedures Online)
http://www.biologicalprocedures.com/bpo/arts/1/20/m20.htm

Quantitative Measurement of mRNA by Competitive RT-PCR. (Springer Protocols)

Improved quantitative real-time RT–PCR for expression profiling of Individual Cells. (Nucleic Acid Research)

Competitive Quantitative RT-PCR (Ambion)

Comparative Study of Different Standardization Concepts in Quantitative Competitive Reverse Transcription-PCR Assays. (JCM)

Procedure for Competitive Quantitative Reverse Transcription PCR. (Transgenomic)

PCR Protocol--Ligation Mediated Suppression PCR

Ligation Mediated Suppression PCR

(adapted from McKinney et al., 1995, Siebert, 1995, Strauss et al., 2001 and Alonso et al., 2003)

PURPOSE: To analyze unknown flanking genomic sequences adjacent to a T-DNA left border

1.) Isolation of DNA
-collect 2-3 young leaves in an eppendorf tube
-add 100 uL extraction buffer and add proteinase K, grind tissue using a blue pestel (no large pieces of leaf should be left).
-add another 100 uL of extraction buffer, vortex, and incubate in 37C for 30 min.
-add 200 uL of saturated phenol and vortex.
-spin at max speed in centrifuge for 2 min.
-collect upper phase to new eppendorf tube.
-add 200 uL of (24:1) chloroform:isoamyl alcohol, vortex, centrifuge at max speed for 2 min.
-collect upper phase into a new eppendorf tube.
-add 18 uL of 3M sodium acetate and add 400 uL of 100% EtOH, mix by inverting and incubate for 10 min at 4C.
-spin in centrifuge at max speed for 10 min.
-pour supernatant off and wash with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour supernatant off and wash again with 500 uL of 70% EtOH
-spin in centrifuge at max speed for 5 min.
-pour off supernatant
-carefully pipette off excess EtOH
-let pellet dry for 45 min in the hood.
-resuspend DNA in 100 uL of TE
-store in -20C


2.) Digestion
-mix together:
50 uL gDNA (from above)
10 uL 10x Buffer 2
1 uL Hind III
39 uL dH2O
TOTAL volume 100 uL

-digest overnight at 37C


3.) Digestion Clean Up
-heat inactivate at 65C for 20 min.
-add 100 µL of chloroform
-mix by inverting tubes
-spin in centrifuge at max speed for 5 min.
-collect upper phase into a new eppendorf tube with 200 uL of isopropanol
-mix by inverting tubes
-incubate at room temperature for 10 min
-spin at max speed for 10 min
-remove supernatant
-wash with 100 uL of 70% EtOH
-spin at max speed for 5 min
-remove supernatant
-dry in hood for 45 min.
-resuspend in 20 uL of dH2O


4.) Constructing adapters for ligation
*adapters for ligation to Hind III ends are made by annealing oligos ADAPS-E1(5’-aattcacctgcccgg/3AmMc7/-3’) w/ a 3’ amino terminal end and ADAPL-E1(5’-ctaatacgactcactatagggctcgagcggccgcccgggcaggtg-3’). Oligos may be purchased from IDT @ www.idtdna.com

-dilute ADAPS and ADAPL to 100uM
-combine in equal amts of ADAPS and ADAPL (i.e. add 10 µL of ADAPS add 10 uL of ADAPL)
-vortex briefly
-place tube in 500 mL of boiling H2O for 2 min
-remove heat and let bath cool for 1 hr. (this is to ensure correct nucleotide pairing)
-store adapter at -20C


5.) Construction of Adapter Library (ligate adapter to digestion)
-mix together:
10 uL cleaned gDNA digestion
1 uL Adapter (100uM)
2 uL T4 ligase (NEB product)
2 uL 10 x T4 ligase buffer (NEB buffer)
5 uL dH2O
TOTAL volume 20 uL
-vortex and incubate at 16C overnight in thermocycler
-heat inactivate at 65 for 20 min
-add 180 uL of TE (this is your adapter library store at -20C)



6.) Primers for 1º and 2º PCR

*Primary products are generated from amplifying primers AP1 (5’-ggatcctaatacgactcactataggc-3’) and PgwLat52LB-WP1 (5’-ctatgttactagatcgaccgg-3’).

*Secondary products are generated by diluting primary products by 50 fold and amplifying with primers AP2 (5’-tatagggctcgagcggccg-3’) and PgwLat52LB-WP2 (5’-caattcggcgttaattcagtac-3’).

-Primers come from IDT and must be diluted to 10 uM concentration before using.



7.) Primary PCR

-mix together:
0.125 uL Ex Taq (Takara)
2.5 uL 10 x Ex Taq Buffer
2.0 uL dNTP Mix
1.0 uL AP1
1.0 uL WP1
17.375 uL dH20
1.0 uL Adapter Library
TOTAL volume 25 µL

*Run on LMS_PCR2 (conditions recommended by Takara)
1.) incubate at 94C for 2 min
2.) incubate at 94C for 30 sec
3.) incubate at 55C for 30 sec
4.) incubate at 72C for 1 min
5.) recycle to step 2 for 29 more times
6.) incubate at 65C for 10 min
7.) hold at 4C forever


8.) Secondary PCR
*Dilute primary PCR:
-98 uL of dH20
-2 uL of primary PCR

*Rxn is setup exactly like primary PCR.


9.) Run on 1% Agarose Gel
5g Agarose Low
500mL 1 x TAE
50uL EtBr

*Always run a ladder to verify our bands are between 200 and 2000bp. Should only sequence the lines that give a clear band.


10.)Sequencing
*Need to clean up PCR rxn using Qiagen or Eppendorf kits. Then setup the rxn to be sequenced.

- mix together:
2 uL cleaned secondary PCR
1 uL secondary primer (WP2)
17 uL dH20
TOTAL volume 20 uL

*Sequencing is done through UNC Lineberger Comprehensive Cancer Center (located on campus), and normal turn around time can range from 1-7days. To access sequences go to http://152.19.68.152/gafsite/Main.asp (listed as UNC-CH Genome Analysis under favorites).

Run sequences through Signal website http://signal.salk.edu/cgi-bin/tdnaexpress.

Unmapped lines can either be digested with a different enzyme (EcoRI),can try sequencing using PgwLat52RB primers, or try TAIL PCR.
*If you use EcoRI you must use the EcoRI adapter and then proceed as normal


Special Notes:
After secondary (nested) PCR, only lines with clear bands are sequenced. Bands are typically between 200 and 2000bp. Lines resulting in a smear or no bands will not provide good sequence. Not all lines with distinct bands will provide good sequence either (some bands are likely artifacts).

In our hands, LB mapping of EcoRI digests has up to 75% success rate. The remaining lines can be mapped by a combination of RB-mapping (with appropriate RB primers), digesting with a different enzyme (i.e. HindIII) (with appropriate Adapter modifications), or TAIL PCR.

The WP1 and WP2 primers described above are specific to pBI121 and vectors with pBI121 left border regions. Similar primers can be designed to sequence from right borders. The nested primer (WP2) should be ~50 bp inside the T-DNA region to accommodate the possibility of border truncation.

Multiple inserts complicate the results. It is possible that each independent insert could result in a distinct nested PCR band. Bands can be excised and sequenced. It is possible to get sequence from both bands, however, by directly sequencing the nested PCR products.


Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653-657

Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA (1995) An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res 23: 1087-1088

PCR Protocol--Differential Display PCR

Differential Display PCR

About Differential Display PCR: All living organisms have thousands to tens of thousands of unique genes encoded in their genome, of which only a small fraction, perhaps 15%, are expressed in any individual cell. Therefore, it is the temporal and spatial regulation in gene expression that determines life processes. The course of normal cellular development as well as pathological changes that arise in diseases such as cancer are all believed to be driven by changes in gene expression. A pressing problem is to identify and characterize those genes that are differentially expressed in order to understand the molecular nature of disease state and subsequently, to devise rational therapies. Differential Display was invented in 1992 by Drs. Arthur Pardee and Peng Liang to allow rapid, accurate and sensitive detection of altered gene expression (Science. 1992, 257:967; U.S. Patent 5,262,311).

Further readings about differential display technique
What's Differential Display (GenHunter)Introduction to differential display technique
Differential Display (Chun-Ming Liu)The following procedures are described:
RNA extraction and qualification
DNase treatment of RNA sample
Reverse transcription
PCR amplification
Separation on acrylamide gels
DNA extraction from bands of interest
Re-amplification by PCR
Separation on agarose gels
Excision of amplified products
Differential Display (Breeden Lab)Detailed protocol for differential display
Differential Display (Plant Molecular Biolgy Lab)It's for plant RNA display. The protocol should be general.

Differential Display-Reverse Transcription-PCR (Gerard Lazo)

Rational primer design greatly improves differential display-PCR.

Applications of Differential-Display Reverse Transcription-PCR.

Differential Display-PCR -- Sambrook and Russell.

Development and optimization of a fluorescent differential display.

The Science Advisory Board – Differential Display PCR.

Perspective: Micoarrays and Differential Display PCR.

Laboratory Techniques: Differential Display - Polymerase Chain Reaction
Effect of Primer Purity on the Banding Patterns of DifferentialDisplay Polymerase Chain Reaction
Differential Display of RNAs (Breeden Lab.)