Clontech PT3139-1 Manual do utilizador - Página 4

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CLONTECH Laboratories, Inc.

I. Introduction continued

transformants provides a comparative measure of PCR fidelity. The fidelity of

Advantage-HF was confirmed by sequencing (Table I). The high level of fidelity

delivered by the Advantage-HF system increases confidence in sequence de-

rived from PCR products and is beneficial in a variety of PCR applications,

including expression studies of amplified full-length cDNAs, generation of cDNA

libraries, RACE, and analysis of homologous genes amplified with degenerate

primers.

TABLE I. FIDELITY OF ADVANTAGE-HF BASED ON SEQUENCING DATA

determined with individual clones after 25 PCR cycles

agrees with published data (Ling et al. , 1991; Cariello et al. , 1991)

High-fidelity amplification of cDNA and Genomic templates

Advantage-HF was used to amplify several cDNA templates of different lengths

(Figure 2). Although amplification of the longest template yielded a reduced

amount of product (Lane 6), this amplified product contains a higher percentage

of accurate copies—nearly 6-fold higher than Advantage, and 20-fold higher than

Taq , according to Figure 1.

Figure 2. Advantage-HF amplification of cDNA fragments. Several fragments were amplified from

Human Placenta cDNA under standard (Lanes 1, 3 & 5) and high-fidelity PCR conditions (Advantage-

HF; Lanes 2, 4 & 6). M: λ/ Hin d III DNA size markers. Lanes 1 & 2: 0.5-kb fragment of glycerol 3-

phosphate dehydrogenase gene. Lanes 3 & 4: 1.3-kb fragment of transferrin receptor gene. Lanes 5

& 6: 2.5-kb fragment of lactoferrin gene. Cycling parameters: 30 sec at 94

5 min at 68

C); 5 min at 68

page

Protocol # PT3139-1

Version # PR76834

Enzyme

Taq

Advantage-HF

M 1

4.4-

2.0-

0.56-

Technical Support TEL:415-424-8222 or 800-662-CLON

Error rate

(per 100,000 bp)

180

2.4

FAX: 415-424-1064 or 800-424-1350

C; 30 x (30 sec at 94