Simplification of genotyping and mutation scanning

Four alternative designs for genotyping by melting analysis are shown in Figure 9.4. Conventional HybProbe® technology requires two probes, each with a single fluorescent label (Bernard et al., 1998). Single HybProbes® (also known as SimpleProbes®) only require one probe with a single label (Crockett and Wittwer, 2001). Unlabeled probe genotyping depends on

Number required d i Derivative of

Probes Labels g melting curve

Number required d i Derivative of

Probes Labels g melting curve

A Î

JJL

A Î

/ \ j ^

G

One None

Unlabeled Probe (LCGreen dye)

c-1

None None

Amplicon Melting (LCGreen dye)

C

Four different methods of SNP typing by melting. Shown for each method are the number of probes and fluorescent labels required, the hybridization design, and the negative derivative of the resultant melting curve. The melting curves of each method (dF/dT vs temperature) show plots of wild-type (long dashes), heterozygous (solid lines), homozygous mutant (dots), and no template controls (grey dashes). The top two methods use covalently-attached fluorescent labels (depicted by open and closed circles), whereas the bottom two methods use a saturating DNA dye.

saturation dyes for fluorescence with no covalent fluorescent labels. Finally, genotyping by amplicon melting also relies on saturation dyes and requires no probes.

Closed-tube genotyping methods that use melting analysis are less complex than allele specific alternatives that require one probe for each allele analyzed (Table 9.2). Melting techniques also have the capacity to scan for unexpected variants under the probes and/or within amplicons. Allele discrimination by Tm (Wittwer et al., 2001) or curve shape (Wittwer et al., 2003, Graham et al., 2005) are promising alternatives to fluorescent color. Conventional closed-tube genotyping methods require expensive fluorescent labels. Conventional scanning techniques, such as SSCP, DGGE, TGGE, heteroduplex analysis, DHPLC, TGCE, and chemical or enzymatic mismatch detection methods require processing and separation steps after

Table 9.2 Comparison of closed-tube, homogeneous SNP genotyping and scanning methods

Method (references)

Probes/SNP genotype

Modifications/ probe

Scanning region

Amplicon melting (Gundry et al., 2003) (Wittwer et al., 2003) (Liew et al., 2004) (Graham et al., 2005)

0

0

Between primers

Unlabeled probe (Zhou et al., 2004a)

1

0

Between primers and within probes

Simple probe

(Crockett and Wittwer, 2001)

1

1

Within probe

Hybridization probes (Bernard et al., 1998)

2

1

Within probes

TaqMan® (Lee et al., 1993)

2

2

(Marras et al., 2003)

2

2

(Whitcombe et al., 1999)

2

3

None

MGB TaqMan® (de Kok et al., 2002)

2

3

None

PCR (Wittwer and Kusukawa, 2005). Closed-tube genotyping and scanning with LCGreen™ dyes are attractive because: 1) there are no processing or separation steps after the initiation of PCR, 2) only standard PCR reagents, unlabeled oligonucleotides, and a saturating DNA dye are required, and 3) analysis is rapid (1-10 min after PCR).

0 0

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