Clontech
login | register | order now | view cart | feedback
Products
PCR


Products >  PCR >  Resources >  Application_Notes >  Direct_PCR_from_Food

TECH NOTE

Amplifying a Gene of Interest from Raw and Processed Meat Samples

Terra PCR Direct outperforms other enzymes with samples containing higher levels of PCR inhibitors

Overview

Terra PCR Direct Polymerase Mix contains an optimized DNA polymerase that allows for direct amplification from a tissue source or crude extracts, without the need for DNA purification. It can amplify DNA targets up to 4 kb as well as difficult templates (including those with a GC-content of greater than 70%)—and it enables DNA amplification in the presence of PCR inhibitors, growth serum, and other source material components.

Comparison with Other PCR Enzymes

Terra PCR Direct Polymerase demonstrates an exceptional ability to provide consistently strong amplification of a target gene (cox1) directly from crude samples–specifically, from different types of raw meat (beef, chicken, and pork) and processed meat (chicken dumplings, beef meatloaf, and roast pork)—when compared to other PCR enzymes (TaKaRa Ex Taq Hot-Start Version [HS] and PrimeSTAR HS, GXL, and MAX).

Terra PCR Direct Polymerase Mix (Lanes 1) shows strong amplification with all six sample types, including the raw beef sample (Panel A). Although the PrimeSTAR series of PCR enzymes (Lanes 3–6) shows good overall performance with the crude extracts in Panels B–F, none of the enzymes other than Terra are able to effectively amplify the raw beef sample (Panel A).

Comparing Terra PCR Direct to other PCR enzymes for amplification from crude samples.

Comparing the ability of Terra PCR Direct Polymerase Mix to amplify a cox1 target gene of interest from different raw and processed meat samples to that of other PCR enzymes—using agarose gel electrophoresis. Lane 1: Terra PCR Direct Polymerase Mix. Lane 2: TaKaRa Ex Taq HS DNA Polymerase. Lane 3: PrimeSTAR HS DNA Polymerase. Lane 4: PrimeSTAR GXL DNA Polymerase, standard protocol. Lane 5: PrimeSTAR GXL DNA Polymerase, high-speed protocol. Lane 6: PrimeSTAR Max DNA Polymerase. Lane M: pHY Marker.

Summary

This experiment demonstrates that most meat extracts can be amplified by TaKaRa Ex Taq HS and PrimeSTAR HS, GXL, and MAX DNA Polymerases, but Terra PCR Direct Polymerase Mix shows a higher tolerance to PCR inhibitors than the other enzymes, regardless of the type of sample used. Terra PCR Direct Polymerase generates a strong amplification band for the cox1 gene from the crude raw beef extract, a rich source of iron, zinc and other metal ions that can interfere with PCR reactions. The fact that the other polymerases showed little or no amplification of the raw beef sample suggests that Terra is the best choice for analyzing meat extracts, as well as other samples that contain high levels of PCR inhibitors.

Methods

Alkali and heat were used to prepare crude DNA extracts from raw meats (beef, chicken, and pork) and processed meats (chicken dumplings, beef meatloaf, and roast pork). The extracts were prepared by heating the meat samples for 10 minutes at 95°C in the presence of sodium hydroxide, followed by neutralization and centrifugation. Various PCR enzymes were used to amplify a cox1 gene fragment from a 1 μl aliquot of each crude extract in a 20 μl reaction volume. After amplification, 3 μl of each reaction was analyzed on a 1% Agarose L03 Gel to compare the effectiveness of each enzyme in amplifying the different crude extracts.

Buy now  >>

Download PDF >>

Top of page

Takara Bio USA is a Takara Bio Company © 2017 Privacy Policy | Terms & Conditions | Terms of Use