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Front Matter Pages Pages Yu Shao, Berta E. Warman, John P.

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Spellman, Kwan H. Cho, Walter C. Low, Walter A. Alan L. Lindstrom, Christopher A. Immunotoxin Treatment of Brain Tumors. Elise A. This is most likely attributable to biased PCR ampli- fication of the alleles. In the given equation, a difference in peak height between alleles is normalized using heterozygous individu- als. The peak height ratio of the heterozygote is also used as a mea- sure of the reproducibility of quantification by SSCP, and should be checked before the determination of actual allele frequencies using pooled DNA. Orita, M.

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Genomics 5, Hayashi, K. Human Mutation 2, Inazuka, M. Marth, G. Sasaki, T. Nucleic Acids Res. Ren, J. Introduction Several large databases are now available which contain infor- mation on hundreds of thousands of single nucleotide polymor- phisms SNPs distributed throughout the genome. Although these databases represent a tremendous resource for studies of human variation and disease, two challenges remain. First is the develop- ment of novel strategies of genotyping known SNP based genetic markers that will allow accurate and rapid high-throughput analy- ses 1,2.

Second is the development of sensitive and specific meth- ods for the detection of previously unknown, novel SNPs in particular genes or genomic regions of special interest. The various methods currently available for detection of SNPs all depend on the ability to detect different physical properties in DNA molecules that result from variations in the nucleotide sequence. These properties include minor differences in thermal melting profiles of two DNA molecules differing in sequence by a single base or structural dis- tortions in perfectly double stranded nucleic acid molecules due to the presence of unpaired or mismatched bases.

However, NMR and X-ray diffraction methods require highly sophisticated instrumentation and can not be easily accessible to every research laboratory for individual genotyping projects. A different set of methods explore the possibility that single base differences in DNA sequences can be detected by differential migration of single stranded molecules containing variant DNA sequences SSCP or double stranded molecules consisting of het- eroduplexes and homoduplexes in electrophoretic gels CSGE.

These methods require minimal manipulation of the PCR amplified genetic materials and are very useful in terms of easy access, cost and high throughput in scanning of large regions of genomic DNA or cDNA for presence of sequence variation 5. Single-Strand Conformation Polymorphism Analysis In recent years, single-strand conformation polymorphism SSCP has been one of the most frequently used methods for iden- tifying single base mutations in many putative disease causing genes 6.

In this method the PCR products are denatured followed by rapid cooling such that the complimentary DNA strands fold back on themselves and acquire specific secondary structures defined by the nucleotide sequence of the fragment to be analyzed. When these fragments are analyzed in a nondenaturing polyacrylamide gel, dif- ferential migration is observed for complimentary strands of the same DNA molecule containing sequence variation as small as a single base.

This method has found its biggest application in rapid and preliminary survey of large sets of samples to determine a rea- sonable estimate of the frequency of a previously known mutation or polymorphism. The use of radioactivity enhances the sensitivity of this method but non-radioactive detection methods can be used Conformation-Sensitive Gel Electrophoresis 49 as well that include fluorescently labeled DNA 7 and silver stain- ing 8. Usually one has to test at least four different combinations of gel electrophoresis conditions including gel temperature room temperature or colder and the presence or absence of glycerol to be certain that the method is not giving any false negative or positive signal.

During PCR, the complimentary strands of the amplified DNA molecules undergo repeated cycles of denaturation and renaturation. Heteroduplexes are formed in the presence of two distinct alleles of a DNA sequence. The generation of heteroduplexes containing looped out bases on one strand due to deletions or insertions were initially observed to give rise to aberrant electrophoretic migration when analyzed on regular non-denaturing poly aery lamide or agarose gels.

The presence of such aberrantly migrating bands was first reported as PCR artifact 9. Thereafter analysis of heteroduplexes by poly- acrylamide gel electrophoresis became very common with best results coming from DNA loops or bubbles of three base pairs or larger.

1. Introduction

White et al. In developing this method, the main emphasis was on the design of a method that is easy to use with standard labora- tory equipment and reagents. Since the method is applicable to PCR products directly, it maximizes throughput and is highly efficient. Thereby, the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis can be enhanced. The method of CSGE uses a non-denaturing polyacrylamide gel with the following modifications: a the crosslinker is 1,4-Bis acryloylpiperazine BAP instead of the traditional bis-acrylamide. The CSGE method has now been applied to analyses of a number of genes.

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All of these genes are large with multiple exons and many novel mutations and SNPs have been identified Reagents 1. Ethylene glycol Sigma, MO , stored at room temperature. Ammonium persulphate Amresco, OH , made fresh daily. Ethidium bromide solution Sigma, MO , stored in the dark at room temperature see Note 2. Conformation-Sensitive Gel Electrophoresis 51 2. Equipment 1. Glass plates 43 cm X 36 cm. Power Supply units that can operate at constant voltage or constant wattage condition.

Large tray for holding CSGE gel during ethidium bromide staining. Whatman 3 MM paper. UV-Transilluminator with large photodocumentation area. Kodak MP4 polaroid camera. Polaroid film type X-Ray autoradiograph films. Solutions and Buffers 1. Running gel buffer 0. Staining solution: 0.

The glass plates for the gel, spacer, and the combs are cleaned every time before the assembly of the gel cassette. Hot water is the best cleaning agent followed by a rinse with deionized water see Note 4. The glass plates, spacers and comb are wiped clean with ethanol and dried with lint free tissue paper. Silanize one of the glass plates to allow easy disassembly of the cassette at the end of the electrophoretic run. Prepare mL of gel solution by mixing Filter and degas the acrylamide gel mixture for 20 min by vacuum filtration using a Nalgene 0. Add 1. Pour gel immediately.

Remove small bubbles formed during this pro- cess by slight tapping on the glass plates. Allow the gel to polymerize for at least 2 h after casting the gel. The use of a high fidelity Taq polymerase such as HiFidelity Taq Polymerase Boehringer Mannheim, IN is recommended to ensure elimination of errors due to incorporation of wrong bases during amplification. Gel Electrophoresis 1. Pre-Run gel at volts for 15 min. Wash sample wells with 0. Conformation-Sensitive Gel Electrophoresis 53 3. Load J. L of sample into each well see Note Run samples at volts for 16 h.

Monitor gel temperature by using thermometer strips C. Scientific, CA.

Staining 1. At the end of electrophoresis, disassemble the gel cassette so that the gel is left attached to one of the glass plates while the other plate is removed. Stain the gel by layering just enough of the staining solution to cover the top surface of the gel for 5 min. The gel surface should be perfectly horizontal such that the thin film of staining solution does not flow out.

After 5 min, destain the gel in distilled water for 10 min. Photographic Documentation 1.

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Cut the relevant section of the gel with a scalpel and lift the gel sec- tion with a piece of dry Whatman 3 MM blotting paper. Release the gel section on the transilluminator by wetting the filter paper with water. Photodocument the ethidium bromide stained bands under transillu- mination with an orange-red color correction filter using Polaroid type film.

Mix 1. L 10X polynucle- otide kinase PNK buffer, 2. Add 2. L genomic DNA 20 ng to a mixture containing 2. L 10X PCR buffer, 3. L labeled primer, 0. L Taq polymerase, and 9. Thermal cycle using the usual PCR conditions. Add 5. L of loading buffer to each PCR tube. Load 4. L of each sample on gel. Store the rest in the plastic beta blocking box in the freezer. Due to higher sensitivity of autoradigraphy, a thinner CSGE gel matrix 0. After electrophoresis, the gel is dried on to Whatman 3MM filter paper and exposed to X-ray films for autoradiography. Gloves should always be worn when working with unpolymerized acrylamide solution.

Ethidium bromide is a carcinogen. Gloves should always be worn when working with ethidium bromide solution. The presence of trace amounts of detergent on the glass plates can lead to smearing of the bands and can significantly disturb the reso- Conformation-Sensitive Gel Electrophoresis 55 lution. Therefore, it is imperative that the glass plates are washed meticulously and rinsed with hot water very carefully to remove any trace amounts of detergent. During each cycle of PCR, amplified DNA molecules undergo denaturation and renaturation and generate homoduplex as well as heteroduplex molecules.

However as the molar concentration of the amplified products increase in the later cycles, complete denaturation of products amplified in previous cycles may not happen. Therefore, some times it may be necessary to dilute the PCR products by a factor of 2 to ensure optimal denaturation and renaturation to favor heteroduplex formation.

The sequence context of a SNP clearly has an important effect on ease of detection by any physical, chemical or enzymatic method. Many other observations suggest that as many as 5 nucleotides flanking a base mismatch may have an influence on the conformational change induced by the mismatch. Hence, it may be necessary to test as many as 4 10 or over a million sequence con- texts to ensure that a given technique can detect all possible mis- matches. Thus, sequence context and nature of mismatch can modify degree of resolution. These CSGE conditions can always be modified to optimize the separation of any known SNP-bearing heteroduplex from the corresponding homoduplex molecules.

In modifying the CSGE conditions, the following factors may be considered. First, optimal resolution of heteroduplex from homoduplex molecules can be obtained for fragments base pair in size. Second, centrally located mismatches are detected more easily than when located within 50 base pairs of either end of PCR product and can be missed 5. The wells should be rinsed every time before loading sample to ensure that the starting front is very uniform — the degree of resolu- 56 Ganguly tion depends to a large extent on shape of the starting sample front. Also individual PCR products can be pooled or loaded onto the same wells at definite time intervals 15 min is an optimum time difference.

The resolution of the heteroduplex bands from the homoduplex bands remains the same whether loaded in batch or individually. The sensitivity of detection can be a function of various factors induced by the experiment. For example, loading too little or too much DNA can mask the heteroduplex band.

This is determined by the amount of starting concentration of PCR products, GC-content of the DNA sequence, as well as relative migration of the two hetero- duplex molecules with respect to the homoduplex molecules. It has been observed that for a particular SNP, complimentary heterodu- plex molecules can have very different migration pattern with one of the two comigrating with the homoduplex molecules. Furthermore, the number of distinct electrophoretically migrating homoduplex and heteroduplex bands can be as large as four representing two wild-type and mutant homoduplex molecules as well as two heteroduplex molecules.

In contrast there can be just two bands where two homoduplex mol- ecules comigrate as well as two heteroduplex molecules comigrate but distinct from the latter species. UV light is damaging for the eyes and skin. Gray, I. C, Campbell, D. Human Mol. Conformation-Sensitive Gel Electrophoresis 57 2. Buetow, K. Shakked, Z. Woodson, S. Biopolymers 28 6 , Ganguly, A.

USA May 24; 91 11 ]. USA 90 21 , 10,, Hayashi, S. Gonen, D. Psychiatry 4 4 , Oto, M. Nagamine, C. White, M. Genomics 12 2 , Williams, C. Genetics 4 2 , Aradhya, S. Human Genet. Liu, W. Has, C, Bruckner-Tuderman, L. Melkoniemi, M. Bignell, G. Finnila, S. Korkko, J. USA 95 4 , Cotton 1. The scientific background of CCM stems from the initial study of sequencing technique 2 in conjunction with other advanced stud- ies associated with thermodynamics and secondary structures of single base pair mismatched DNA or RNA 3.

Such literature data confirmed that the mismatch point is locally destabilized and highly susceptible to many enzymatic 4,5 and chemical reactions 6. Based on this platform, the CCM technology theoretically estab- lishes the simplest chemical means to detect mismatch and thus mutation at this point in time. The method employs two commer- cially available chemicals, hydroxylamine 7 and potassium per- manganate to react with unmatched cytosine and thymine, respectively.

The modification of the mismatch is then followed by From: Methods in Molecular Biology, vol. Since the first protocol was described in 11 , the performance of this method has been continuously improved and the present protocol has some major advantages: 1 potassium permanganate KMn0 4 has replaced the toxic osmium tetroxide Os0 4 9,10 ; 2 if both mutant and wild-type DNA samples are labeled a double chance of mutation detection occurs; 3 the method is sensitive to as low as 0.

Other alternative versions for mismatch detection have been established on the basis of enzymatic cleavage 4,5. These meth- ods are out of the scope of this protocol and they are briefly dis- cussed for comparative purposes see Note 1. If the two sequences mutant and wild-type are different at any oligo- nucleotide base, a complementary pair of single base pair mis- matches will be generated and mismatched C and T bases will be susceptible to chemical modification and cleavage.

How- ever, to obtain two chances of detecting a mutation, labeling of both wild-type and mutant DNA is recommended see Note 2 and Fig. Materials 1. Store the TE buffer at room temperature. Solid-phase chemical cleavage of mismatch. Both perfect and mismatch duplexes are immobilized on silica beads. Chemical modifica- tion reactions are carried out while DNA duplexes still remain on solid support. One sample of DNA is treated with hydroxylamine and another with potassium permanganate. Piperidine treatment simultaneously cleaves the mismatched point and releases the samples for gel electro- phoretic analysis.

Water is added to adjust the final volume to 4 mL. Prepare the solution freshly before use. Arrows represent cleavage at mismatched T and C bases. Cleavage-dye solution: Add 20 0. L dye 50 mg blue dex- tran [Aldrich] per mL. La Jolla, CA. Detection of DNA Mutations 63 3. Methods The assay should be carried out in a fume hood as hydroxylamine, acrylamide, formamide, and piperidine are noxious chemicals. DNA Preparation 1. Amplify plasmid DNA about 0. Purify the resulting DNA samples wild-type and mutant by using the purification kit Stratagene; see Note 3 or cutting a band from an agarose gel.

Formation of Heteroduplex DNA 1. Place 1 uL of heteroduplex DNA samples 0. Place 1 uL of homoduplex DNA samples 0. Gently mix the tubes on shaker at room temperature for h. Reaction with KMn0 4 1. Centrifuge the tubes at g and carefully decant the supernatant by Pasteur pipet. Wash the pellet twice with 0. L Ultra-wash solution. Dry the beads in open air for 15 min. Reaction with Hydroxyiamine 1. Add 30 iL of 4. Centrifuge the tube and carefully decant the supernatant. L of Ultra-wash solution. Cleavage by Piperidine 1.

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Add 10 J. L of cleavage dye solution to all four reaction tubes. Cool the tubes on ice and the solid beads are separated by centrifuge see Note Load the supernatant on to a denaturing polyacrylamide gel 4. Electrophoresis will take approx 3 h for analysis of a bp fragment. Detection of DNA Mutations 65 3.

Result Analysis Mismatch detection is based on comparative study between traces for homoduplex and heteroduplex DNA samples. Cleavage peaks present in the trace of heteroduplex sample but not the control homo represent the mutation. For typical example, see Figs. The major advantages of the EMC are: 1 the enzyme binds and cleaves at the mismatched point in a one-step reaction while the CCM requires two-step process; 2 one enzyme can recognize all types of mismatches, whereas the CCM requires two types of chemicals to achieve the same purpose. However, the EMC is more expensive and often suffers from the need to optimize thoroughly the reaction conditions, and substantial cleavage of matched bases occurs leading to high background bands.

The ribo- nuclease method needs the production of RNA to form the duplexes, but as the cleavage at the mismatches is double-stranded it can be analyzed on an agarose gel. A kit is available from Ambion USA. False-positives and -negatives have not been reported so far. How- ever, labeling both mutant and wild-type DNA will offer two chances of mutation detection in the event of the rare occurrence of unreac- tive mismatch. Radioactive labels can also be used instead of fluo- rescent ones.

PCR products of mutant and wildtype DNA can be conveniently purified by using the Stratagene purification kit or by agarose gel- electrophoresis. In the latter case, the band is precisely cut and loaded on to silica beads. The control trace homoduplex, top shows no cleavage peak and the mismatched DNA trace heteroduplex, bottom displays a strong cleavage peak of the mismatch T base in the 5' FAM sequence. Note consistent background in both traces, which is essentially a chemical sequencing trace of T bases allowing confidence that reaction has occurred and a position reference.

The control trace homoduplex, top shows no cleavage peak and the mismatched DNA trace heteroduplex, bottom displays a strong cleavage peak of 3'HEX sequence at the mismatched C base. Note this is the second chance of detecting the mutation, the first being in Fig 3. Size, concentration, and temperature of the melting heteroduplex for- mation should be taken into account as there can be poor heterodu- plex formation in some cases. A test should be carried out on agarose gel to make sure that no intense multiple bands or smeary bands are present after heteroduplex formation.

Attachment of homo- and heteroduplex DNA onto the commercially available silica beads are the first important step in solid-phase CCM method. The adsorption is achieved under relatively high concentra- tion 3 M of TEAC salt solution and the DNA molecule remains attached throughout the modification and washing steps. The DNA length is limited by the analytical technique, fidelity of the heteroduplex formation, and the solid supports. In our study, the silica beads and special conditions are most suitable for up to bp fragments of DNA. Refer to liquid-phase protocol for larger frag- ments kb In principle, bases other than TEAC e.

Aqueous KMn0 4 solution should be freshly made before use. The aging solution after 1 d turns brown-yellow with precipitation of Mn0 2. The reaction is dependent on temperature and concentration of substrates. Usually the concentration of chemical given is correct for approx ng of total weight of DNA.

Prolonged incubation can lead to overreaction and destruction of the heteroduplexes and fragment DNA. Underincubation can give rise to no cleavage bands. Time courses of incubation are recommended when starting to use this test. Both mismatch cleavage and release of DNA from silica beads are achieved in one-step reaction. Separation of excess piperidine prior to the gel electrophoresis step is not required. Formation of DNA homo- and heteroduplexes were performed under the standard conditions and subjected to the solid-phase CCM procedure as described ear- lier.

The results of the cleavages are shown after electrophoresis and analysis on an ABI sequencer. Figures 3 and 4 show single and strong cleavage peaks as the result of cleavage reaction of T and C mismatches by KMn0 4 see Fig. Ellis, T. Maxam, A. USA 74, Kennard, O. Youil, R. Myers, R. Smooker, P. Mutation Res. Cotton, R. Gogos, J. Roberts, E. Lambrinakos, A. USA 85, Introduction DNA sequencing, while relatively laborious, is the gold standard in mutation detection and single nucleotide polymorphism SNP discovery. The most widely used approach is direct DNA sequenc- ing of polymerase chain reaction PCR products with dye-termina- tor chemistry analyzed on automated DNA sequencers 1.

Although the quality of DNA sequencing data has improved signifi- cantly over the last few years, the peak pattern remains uneven and random artifacts are seen from time to time 2. Because human cells are diploid, DNA sequence of a heterozygote contains a locus where two different bases occupy the same site. The uneven peak pattern makes it difficult sometimes to discern these composite peaks because one of the two polymorphic bases may be dispropor- tionately smaller than the other base and the base-calling algorithm of the automatic DNA sequencer misses the correct call Fortunately, the peak pattern of a DNA sequence is highly repro- ducible and is determined by the local sequence context Therefore, when the DNA sequencing traces of multiple individuals are compared to each other, the peak patterns of the heterozygotes and the homozygotes are noticeably different and the mutations or polymorphisms can be identified easily 5.

In addition, the relative peak heights of the polymorphic bases can be used to estimate allele frequencies when pooled DNA samples are sequenced and compared to a reference DNA sequence. Accordingly, PCR primer design that emphasizes specificity and high yield is of great importance. A good primer design program is the modified Primer3 6,7.

If the PCR primers selected are found to be unique in the genome by homology searches against the human genome DNA sequence, the chances of obtaining a specific PCR product are high. When the desired PCR product is the only species generated in the PCR reaction, one can use the product directly in the sequencing reaction without purification. The protocol is further simplified by using an asymmetric PCR approach, performing the amplification with a mixture of the PCR primers and a reduced amount of deoxyribonucleotide triphosphates dNTPs.

The excess dNTPs do not interfere with the sequencing reac- tion because the sequencing mix contains a much higher concentra- tion of dNTPs. At the end of the sequencing reaction, the dye-terminators are removed by size-exclusion chromatography using spin columns. Spin-column purification produces much better quality data than those generated by ethanol precipitation.

The part of the sequencing trace with the highest quality is the bp segment between bases from the 3'-end of the sequencing primer. The sequenc- ing data can be made more uniform from sample to sample by "trimming" the low-quality data at the beginning and end of the sequencing traces before reanalyzing the data.

If done the same way for all samples for the same marker, the sequencing traces can be compared more easily. By comparing the peak patterns of sequenc- ing traces from a number of individuals, one can identify differ- ences between them at the polymorphic sites. When homozygotes of different alleles are present among the samples sequenced, the polymorphisms can be identified easily using any sequence alignment programs. In cases where the minor allele frequency is low, one usually sees only homozygotes of one allele and a handful of heterozygotes.

Here, one relies on a break in the peak pattern where the heterozygous samples exhibit a peak whose height is reduced by half when compared to the homozy- gotes together with the telltale sign of a second base underneath and an often observed phenomenon of a change in peak height in the base 3'- to the polymorphic base 5. If equal amounts of genomic DNA from a group of individuals are pooled together, the pooled samples can be amplified and sequenced as usual.

The pooled DNA sequencing trace can be compared against a reference sequencing trace for allele frequency estimation. Reagents 2. PCR primers are designed by modified Primer3 program 6,7. Easy-peel heat-sealing foil Marsh Bio Products. Silicone compression mats Marsh Bio Products. Strip caps Midwest Scientific.

MicroAmp Optical well reaction plates Applied Biosystems. Sequencing 1. Centri-Sep 96 well gel filtration plate, Princeton Separations. Horizontal gel electrophoresis devices. Thermo-sealer Marsh Bio Products. Version 3. PCR Reaction 1. Amplify genomic DNA in 30 J. L reaction mixtures containing 3 uL of genomic DNA 12 ng , 3.

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Prepare 0. Load the entire 36 uL reaction mixture onto the agarose gel. Transfer the gel slices to 1. Add 1 mL resin and mix thoroughly for 20 s. Do not vortex! For each sample prepare one Wizard minicolumn attached to a syringe barrel and insert it to the vacuum manifold, add the DNA- resin mixture to the syringe barrel, and apply vacuum until all liquid passes through minicolumn.

Air dry resin by applying vacuum for an additional 30 s. Remove the syringe barrel and centrifuge the minicolumn at 10,g for 2 min in a 1. Discard the washing. Transfer the minicolumn to a clean 1. Elute DNA by adding 50 0. L ddH 2 to the minicolumn and incubate at room temperature for 1 min; follow this with centrifugation at 10,g for 20 s. Sequencing Reaction 1.

Assemble the sequencing reaction 12 iiL total volume by adding 5 J. L of purified PCR product to a strip tube containing 2. Asymmetric PCR 1. Thermocycling conditions are the same as those found in Subheading 3. For sequencing add 2. Purification of Sequencing Products 1. Add 12 uL of ddH 2 to the 12 uL of sequencing reaction products see Note 5. For a small number of samples, bring the desired number of Centri- Sep 8 strips to room temperature before use.

Remove the top foil and integral bottom of the strips and spin for 2 min at g to remove the storage liquid. Transfer the samples onto the center of the gel bed without disturb- ing the gel surface and place the strips onto clean PCR strip tubes. Collect the samples by centrifugation for 2 min at g. For a large number of samples, use the Centri-Sep Well gel filtra- tion plates purification approach. Bring plates to room temperature and remove adhesive foils from the bottom and then the top. Place the plate on top of a Well wash plate and centrifuge at 1 g for 2 min to remove the storage liquid.

Transfer sequencing reaction mixtures onto individual wells of the plate, taking care that the samples are loaded onto the center of the gel bed without disturbing gel surface. Place the gel plate on top of a clean Well collection plate and cen- trifuge at g for 2 min. SNP Identification Sequence at least two different individuals for sequence compari- son.

The goal is to remove poor quality bases from the sequencing trace and boost sequencing sig- nal the same way for all the sequences being compared. This is done by modifying the "start point" and "end point" of the individual sequence traces and reanalyze them. A Macintosh version is also available from the same company. Compare the peak patterns to each other by looking at the traces and positions flagged by Sequencher. When both alleles of a variation are found in homozy- gotes in the samples sequenced, the Sequencher program will des- ignate the base position as being occupied by an N.

If only one allele is represented by homozygotes while the second allele is only found in heterozygotes, it is less obvious to the computer program and one has to examine every base carefully. However, in the sequencing trace with the heterozygote, the G peak height at the candidate SNP site is about half the size as the homozygous G peak, and it is always accompanied by a second peak underneath and an observable change in peak height in the base 3'- to the polymorphic base.

Allele Frequency Estimation Software packages used are the same with the ones listed in Sub- heading 3. Typically, we prepare pools consisting of individuals. For opti- mal results, we design PCR primers to place the SNP in the middle of the sequencing fragment or at least bp away from the 3 '-end of the primer to be used in sequencing. Trim and reanalyze sequencing traces as described in Subheading 3. Growth of Plants and Preservation of Seeds. Pages Sterile Techniques in Arabidopsis. Control of Pests and Diseases of Arabidopsis.

Establishment and Maintenance of Cell Suspension Cultures. Callus Culture and Regeneration. Protoplast Isolation, Culture, and Regeneration. Preparation of Physiologically Active Chloroplasts from Arabidopsis. Purification of Mitochondria from Arabidopsis. Preparation of DNA from Arabidopsis. Chloroplast DNA Isolation. Preparation of RNA. Genetic Analysis.