C

Fluorescent dye labeled primer

One strand of PCR product is labeled with fluorescent dye allelic ladders that are labeled with the same fluorescent dye so that differences in dye mobilities do not impact allele calls.

SUMMARY OF FLUORESCENCE DETECTION

A laser strikes a fluorophore (dye) that is attached to the end of a DNA fragment. The fluorophore absorbs laser energy and then emits light at a lower energy (higher wavelength). Filters are used to collect only emitted light at a particular wavelength or range of wavelengths. Photomultiplier tubes or charge-couple devices are used to collect and amplify the signal from the fluoro-phore and convert it to an electronic signal. These signals are measured in relative fluorescence units and make up the peaks seen in capillary electro-pherograms or bands on a gel image.

Advantages of fluorescence detection methods include higher sensitivity and a broader dynamic range than comparable colorimetric detection methods (e.g., silver-staining) and the capacity for simultaneous multi-parameter analysis of complex samples such as multiplex PCR products with different fluorescent labels.

FLUORESCENT DYES USED FOR STR ALLELE LABELING

Table 13.2 lists a number of fluorescent dyes that are commonly used to label PCR products for genotyping applications. The chemical names for the dyes are listed as well along with their excitation and emission wavelengths. AmpFlSTR® kits from Applied Biosystems use PCR primers that are labeled with the NHS-ester dyes 5-FAM, JOE, or NED (Applied Biosystems 1998). GenePrint« PowerPlex« 1.1 and 2.1 kits from Promega Corporation use PCR primers labeled with fluorescein and tetramethyl rhodamine (TMR). Newer STR kits, such as Identifiler, include an expanded set of four dyes (6FAM™, VIC®, NED®, and PET«) for labeling PCR products in multiplex amplification reactions.

FAM and fluorescein fluoresces in the blue region of the visible spectrum, JOE in the green region, and NED and TMR in the yellow region. AmpFlSTR® kits utilize a fourth dye named ROX that fluoresces in the red region to label an internal standard for DNA sizing purposes. PowerPlex® STR kits use the red

Table 13.2 Characteristics of commonly used fluorescent dyes in STR kits and other genotyping applications.

dichlorofluorescein

VIC

Proprietary to Applied Biosystems

538

554

NED

Proprietary to Applied Biosystems

546

575

PET

Proprietary to Applied Biosystems

558

595

LIZ

Proprietary to Applied Biosystems

638

655

ROX (CXR)

6-carboxy-X-rhodamine

587

607

Fluorescein (FL)

Fluorescein

490

520

TMR (TAMRA)

N,N,N',N'-tetramethyl-6-carboxyrhodamine

560

583

TET

4,7,2',7'-tetrachloro-6-carboxyfluorescein

522

538

HEX

4,7,2',4',5',7'-hexachloro-6-carboxyfluorescein

535

553

Rhodamine Red

Rhodamine Red™-X (Molecular Probes)

580

590

Texas Red

Texas Red®-X (Molecular Probes)

595

615

SYBR Green (intercalator)

Proprietary to Molecular Probes

497

520

Dye

Chemical Name

Excitation

Emission

Maximum (nm)

Maximum (nm)

5-FAM 5-carboxy fluorescein 493 522

5-FAM 5-carboxy fluorescein 493 522

dichlorofluorescein

Figure 13.3

ABI dyes used in four-color STR detection. The circle portion of the dye highlights the succinimidyl ester, which is used for dye attachment to the fluorescent oligo. In the AmpFlSTR kits, TAMRA has been replaced by NED (also a yellow dye). PowerPlex 1.1 and 2.1 STR kits use fluorescein and TMR, which are very similar to FAM and TAMRA, respectively. The red dye ROX is used to label an internal sizing standard and is the same as CXR used in PowerPlex kits.

FAM (Blue)

C-OH II

C-OH II

JOE (Green)

och3

(ch3)2n

TAMRA (Yellow)

(ch3)2n

-(CH3)2

dye carboxy-X-rhodamine (CXR) for labeling their internal size standard. ROX and CXR are essentially the same dyes (Singer and Johnson 1997).

The 5-FAM, JOE, and NED dyes are fluorescein derivatives that have spectrally resolvable fluorescent spectra. The structures of four commonly used fluorescent dyes from Applied Biosystems are shown in Figure 13.3. Because the structure of NED is proprietary to Applied Biosystems, TAMRA, or tetramethyl rhodamine, has been included in its place for a yellow dye. An additional dye named LIZ® is used to label the internal size standard run with 5-dye detection systems from Applied Biosystems. LIZ emits at ~650 nm and yet still has excellent sensitivity because it is an energy transfer dye (see D.N.A. Box 13.1). With a 5-dye detection system, the four dyes 6-FAM, VIC, NED, and PET are used for labeling PCR products and the fifth dye (LIZ) is used to label the internal size standard.

The fluorescent emission spectra of the four ABI dyes, 5-FAM, JOE, NED, and ROX, are shown in Figure 13.4. Each of the fluorescent dyes emits its maximum fluorescence at a different wavelength. This fact is used to design filters that

The brightness of fluorescence dyes depends on a number of factors including how close a dye's absorbance maximum is to the excitation wavelength of the laser used to excite the dye. If two dyes - a donor and an acceptor - are in close physical proximity to one another and possess overlapping emission and absorbance spectra, then energy is transferred between the two dyes and can improve the amount of energy absorbed by the acceptor dye, which results in an increase in the fluorescence output of the acceptor dye.

Richard Mathies' group at the University of California-Berkeley has pioneered the development of a number of energy transfer (ET) dyes for the past decade to enable more sensitive detection of DNA with brighter dyes. This work influenced the development of the BigDye chemistries used by Applied Biosystems for DNA sequencing and the dye LIZ used in STR typing with 5-dye chemistry kits such as Identifiler™, SEfiler™, and Yfiler™. Note that LIZ has an emission wavelength of approximately 650 nm and yet is still sensitive (i.e., well-excited) with the 488 nm argon ion laser. This type of sensitivity with such a large spread in the excitation and emission wavelengths would not be possible without donor and acceptor energy transfer dyes.

Most ET dye-labeled primers carry a fluorescein derivative at the 5'-end as a common donor since it has an absorbance maximum that is near the 488 nm argon ion laser used in the ABI 310 and other common DNA detection platforms. Other fluorescein and rhodamine derivatives often serve as acceptor dyes and are attached to the primer within a few nucleotides of the donor dye. Efficiency of ET primers can generate 2- to 6-fold greater fluorescent signal than that of the corresponding primers or fragments labeled with single dyes.

Sources:

Ju, J. et al. (1995) Design and synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysis. Analytical Biochemistry, 231, 131-140. Ju, J.Y., Glazer, A.N., Mathies, R.A. (1996) Energy transfer primers: a new fluorescence labeling paradigm for DNA sequencing and analysis. Nature Medicine, 2, 246-249. Rosenblum, B.B. et al. (1997) New dye-labeled terminators for improved DNA sequencing patterns. Nucleic Acids Research, 25, 4500-4504.

Energy transfer dyes capture the signal from each dye. The filters used to separate the various colors are shown as boxes centered on each of the four dye spectra. Note that there is considerable overlapping in color between several of the dyes in the filter regions. Blue and green have an especially high degree of overlap. This overlap influences the degree of 'pull-up' that can occur between two dye detection channels (see Chapter 15).

The dye sets for PowerPlex® kits that are detected on the Hitachi FMBIO II fluorescent scanner (see Chapter 14) are shown in Figure 13.5. Notice that the fluorescence emission spectra for these three dyes have less spectral overlap because they are further apart.

Figure 13.4 Fluorescent emission spectra of ABI dyes used with AmpFlSTR kits. ABI 310 Filter Set F is represented by the four boxes centered on each of the four dye spectra. Each dye filter contains color contributions from adjacent overlapping dyes that must be removed by a matrix deconvolution. The dyes are excited by an argon ion laser, which emits light at 488 and 514.5 nm.

5-FAM JOE NED

Spectra Fam Tamra Sybr Green

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