PERSONAL MAL REMUNERADO

The Wave® method for detecting SNPs involves the analysis of homoduplex and heteroduplex structures that are formed between reference and mutant DNA molecules by ion-pair reversed-phase high performance liquid chromatography under partially denaturing conditions using the Wave® Nucleic Acid Fragment Analysis system. The system has several advantages including; replacement of gel electrophoresis, it confers high sensitivity, allowing screening of a large number of

Kb GG GT GG GT TT TT Control Kb 561bp. 289bp. 240bp. 33bp

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50% cu t G /T 100% cu t T /T F i g u r e 4 . 3 T s p 5 0 9 I R F L P a n a l y s i s .

A gel displaying the Tsp site phenotypes is shown in I. If the enzyme does not cut the template at the restriction site then a G/G phenotype is assigned, a 50% cut is recorded as a G/T and in the T/T phenotype is represented by a 100%

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C A G T G G T A C

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C A G T N G T A T G T AC

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N o c u t T /T F i g u r e 4 . 4 Bt s I R F L P a n a l y s i s . BtsI phenotypes are shown in I. If the enzyme cuts the template 100% at the restriction site a G/G phenotype is assigned, if a 50% cut is observed a heterozygote state is assigned and the enzyme does nt cut a T/T homozygote. Corresponding sequences for the variation are shown in II.

K b TT TT CT CC C o n tr o l Kb 515bp ^ 441 b p ^ 74bp T G T T T G G A G T G T T N G G A G T G T T C G G A G N o e u t T /T 50% e u t C /T 100% e u t C /C

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segregation in Family H Hpy CC c c T C T C

F i g u r e 4.5 Hpy 188 I RFLP anal ysis. The RFLP gel is shown in 1. If the enzyme cuts the template 100% at the restriction site a C/C phenotype is assigned, if a 50% cut is observed a C/T phenotype is assigned and a T/T homozygote does not cut. The sequences corresponding to the variation are displayed in II. The correct segregation of marker Hpy is shown in a b r an ch of t he Fa mi l y H in III.

samples and is ideal for screening for polymorphisms in candidate regions/genes (Liu et a l, 1997). The average processing time per sample is seven minutes allowing for screening of many samples. DHPLC was used as a scanning method for mutation detection in a panel of TSC2 patients and its superiority to SSCP has been acknowledged, particularly for single base substitution mutations (Choy et al, 1999) and has been used in studies of chromosome Y (Underhill et al, 1997). Significant enhancement of sensitivity for this system has been shown by using fluorescent labels (Hecker e/a/., 1999).

There are three modes of operation that can be performed using this technology depending on the temperature at which separation is performed. Under non-denaturing conditions, separation of sequence independent fragments is possible and enables high resolution separation of fragments for various assays. At fully denaturing temperatures, single stranded molecules can be separated for purification purposes and for primer extension genotyping assays. The partially denaturing mode of fragment separation allows a sensitive and accurate method of discrimination which this study employs.

4.2.2.1 Wave® Analysis:partially denaturing mode

A mixture of hetero and/or homo duplexes is formed when a PCR product is hybridized. Samples are denatured at 95°C for 5 minutes and then slowly reannealed by ramping the temperature down to 25°C at a rate o f 0.1 °C /4sec. The DNA strands separate and randomly reanneal to form a mixture o f three species: a mutant homoduplex, a reference and mutant heteroduplex and a reference homoduplex.

Homoduplexes and mismatched heteroduplexes formed during reannealing of the PCR product are easily distinguishable due to the alteration in the structure o f the molecule and are visualized as characteristic patterns o f peaks corresponding to the specific hybridized mixture (Figure 4.6). In the case of SNP genotyping, a heteroduplex typifies the heterozygote state and the two homoduplexes correspond to each o f the two homozygotes (Kuklin et a l, 1997; Taylor et a l, 1998, 2000). The two homoduplexes are distinguishable on the basis o f elution times due to the properties of the DNASep column matrix, and the operation of a gradient which includes acetonitrile as the organic solvent. Thus, DNA variants announce themselves on the basis of physical alterations. Although the system is predominantly used for

wild-type mutant heteroduplexes homooduplexes

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4 5

Retention Time (mln)

Figure 4.6 Schematic of heteroduplex formation for Wave mutation analysis. The PCR products of reference and variant alleles shown in I. differing by one base pair, are denatured and slowly reannealed. The resulting sample contains a mixture of homo- and heteroduplexes; reference and variant homoduplexes melt at higher temperatures than the mismatch heteroduplex comprising of reference/variant sequence. It is this difference in melting temperature that ultimately discriminates and identifies mutations by DHPLC, shown in II (Adapted from Transgenomics).

screening and the identification of mutations in mutant individuals compared to reference sequences, theoretically it is possible to genotype with DHPLC since unique characteristic patterns for individual changes can be detected. The rationality o f using the Wave® system for allele discrimination was also part of the aim for this study.

4.2.2,2 Mechanics o f the Wave® system

A short linear region of single-stranded DNA yielded as a result o f mixing mutant and reference (or mismatch base pairing) during ramped reannealing o f PCR, is the structural alteration that is used to distinguish and separate the heteroduplex from the homoduplex species and ultimately identify the polymorphism.

The separation of heteroduplex and homoduplex species is achieved on the Wave® system using stationary phase alkylated (polystyrene/divinylbenzene) particles that are converted into a dynamic anion-exchanger by an ion-pairing reagent. The trialkyl groups of the positively charged ion pairing reagent, TEAA interact with the hydrophobic surface of the DNASep cartridge. TEAA then acts as a linker interacting electrostatically with the anionic phosphate groups of the oligonucleotide making it a more hydrophobic species.

The positively charged double stranded molecules (homoduplexes) have stronger interactions with the column matrix than the heteroduplex species (as single stranded DNA is retained less strongly on the DNASep Cartridge (Huber and Berti, 1996) and are therefore retained on the column for longer, leading to the separation of two species. TEA ions partially cover the stationary phase, which allows separation of DNA molecules by fragment size. The bridging interaction of the TEA ion is strong but can be modulated with buffers. Separations can be size dependent or size and sequence dependent based on the ion-pairing reagents used.

There is a proportional relationship between the association of TEAA and the binding to the DNA separation column. Dissociation of the TEAA-bound DNA from the column is accomplished by an elution gradient comprising 0.1 M TEAA and 25% acetonitrile. The proportions of reagents are changed over time allowing the elution of DNA; smaller DNA fragments with less TEAA bound are eluted before the larger fragments.

Under partially denaturing conditions heteroduplex DNA start to denature first and elute earlier than homoduplexes; with A-T homoduplex being marginally more

denatured than the C-G homoduplex. It is this marginal difference observed in retention times of the peaks which assists genotyping with respect to the characteristic patterns observed for each duplex species. Providing conditions are precise, the heteroduplex species pattern is observed as separate peaks or occasionally as shoulders on the leading edge of the homoduplex peak. The manner in which a heterozygous peak resolves is dictated by the specific nucleotide mismatch present and the melting characteristics of the flanking nucleotides (discussed in section 4.2.2.3). Separation of homo and hetero peaks are monitored at 260 nm, a wave length at which nucleic acids show a strong absorption.

4.2,23 Prerequisites fo r successful Wave^ analysis

For successful genotyping using the partial dénaturation mode, two important areas must be considered; preparation of the PCR fragment and analysis of samples. Preparation o f PCR reactions requires precise primer design and protocol (section 2.2.1; 2.2.7,2.3). Proof reading Pfu polymerase is also used to minimize PCR induced mutations.

The analysis of samples comprises two important aspects; separation gradient and separation temperature. A melting profile is constructed using the WAVEMAKER'^’^ software which predicts temperature and gradient conditions that would be most suitable to resolve duplexes for a given sequence. Many aspects of a sequence are considered, for example GC rich regions o f an amplicon lose helicality at higher temperatures than AT rich domains.

Theoretical ideal temperature profiles for a fragment are given by WAVEMAKER”^^ in order to determine the most suitable temperature for SNP resolution using DHPLC. Figure 4.7 is an example of the actual temperature profile for SNP N7F indicating that a temperature o f 62°C is most suitable for this fragment. According to the manufacturers guidelines SNPs will resolve optimally if the helical fraction range is between 70% to 85%. The WAVEMAKER™ software predicts the average Tm for the fragment under analysis, the difference between this and the optimal temperature for resolution is also considered and a time shift applied accordingly.

The optimal elution of a fragment is determined by the gradient (WAVEMAKER”^^ software). Figure 4.8 shows the actual gradient profile for the

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Three different txirticl temperature poflles 0 so too 150 ( f i n a l r -T em perature p ip je^ Profile (slow) / ____ I I I 200 250 300 350 400 450 BesePosibon |ê î ParbaFD enatured C |62 PartiaF D enatu nn gl C [63 P artiaF O enatu nn gl r

The best partial temperature (62°C)for the position of interest within the sequence, where percentage helicality is appropriate for detection, between 50 and 85%

Figure 4.7 A Wave temperature profile for marker N7F. The display as seen on the Wave detection system, showing the ideal melting profiles for the fragment N7F (5029 bp). The the ideal temperature at which the appropriate helicality is predicted, thus enabling detection recommended at 62“C. This is achieved by taking into account all the sequence flanking the SNP located at position 194 bp.

Oven temperature Optimal %B at 53%

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AppTjipe Sequence M e irg Giadent 0 Into R W r r ^ ij N5F 279 N5F@59 100 300 -G radient T a b le - 279bpalTm 279bpat50C Tme 2 0 Display %A 509 - 3 lie s 1 "491 i r %c 0 0

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p "o 2 ] G radient Durabon j a s min [Normal ^ C lean Durabon j o T " min [Ô %B Equilibrabon Durabon |o i min |0 5 ~ min

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5 3 0 step Time [% A 1%0 l%c Loading 0 0 52 48 Start Gradient 0 5 47 53 Stop Gradient 5 0 38 62

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Start Equilibrate 5.7 52 48

Stop Equilibrate 5 8 52 48

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wittr run time of smapie

Figure 4.8 Gradient conditions for marker N5F on the Wave analysis system. The gradient applicable for analysis o f fragment N5F (located at 17 729) on the Wave“ analysis system is shown. This shows that an oven temperature o f 59°C and %B at 53% will elute the fragment approximately five minutes after entering the column.

fragment N5F showing the optimal elution of a fragment at an oven temperature of 55°C. The buffers A and B moderate the TEA-DNA bound to the column and dictate the elution times. DNA fragment elution profiles were captured online and visually displayed using the Transgenomic WAVEMAKER'^'^ software. Chromatograms were analysed and multiple controls used to genotype individuals.

4.2.2.4 Site B SNP genotyping using the Wave® system

Primers for several SNPs in Site B and additional SNPs taken from TSC database were designed according to manufacturers instructions for Wave® analysis. The fragments were optimized and tested for their suitability to the Wave® detection system. O f the 12 SNPs two (N5F and N7F) were considered most effective for analysis using the Wave® system due to the suitable characteristics of the sequence. N7F was to be typed strictly using this method whilst N5F was typed in a selection of individuals by different methods for a comparative exercise (section 8.5).

Incorporation o f an unknown sample with a reference type is recommended. This was initially carried out for a few individuals, however due to the large number of individuals to be analysed this was not considered a feasible option. As an alternative, chromatograms of peak patterns and elution of several known sequenced individuals were monitored and thereafter used as controls when genotyping samples.

The heterozygous individuals were identified by the characteristic heteroduplex peak, whilst the homozygote individuals were distinguished on the basis of elution times and slight peak variation between the two homoduplex peaks (note: controls were used for all experimental procedures).

Figures 4.9 and 4.10 display the homozygote and heterozygote Wave® patterns for SNPs N7F and N5F. The associated molecular basis o f the polymorphisms are also shown, together with the correct segregation of N7F and N5F in CEPH families 66 and 884.

In document El retraso en la impartición de justicia laboral por parte de la Junta Local de Conciliación y Arbitraje en el Estado de Puebla de 2010 a 2016 (página 117-120)