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Molecular Methods in Bioremediation

Molecular Methods in Bioremediation Polymerase Chain Reaction (PCR) PCR Machine Gel Electrophoresis Primer Primer sequence Annealing Size Reference temperatures ( C) 16S rDNA universal-F 5 ′ -CCGTCGACAGAGTTTGATCCTGGCTCAG- 3 ′ 55 1500 1 16S rDNA universal-R

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Molecular Methods in Bioremediation

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  1. Molecular Methods in Bioremediation

  2. Polymerase Chain Reaction (PCR)

  3. PCR Machine

  4. Gel Electrophoresis

  5. Primer Primer sequence Annealing Size Reference temperatures (C) 16S rDNA universal-F 5′-CCGTCGACAGAGTTTGATCCTGGCTCAG-3′ 55 1500 1 16S rDNA universal-R 5′-CGGGATCCACCTTGTTACGACTTCACCC-3′ 55 1 Bacillus sp. -R 5′-CCCAGTTTCCAATGACC-3′ 55 640 1 Pseudomonas sp. -R 5′-CCTTCCTCCCAACTT-3′ 50 440 2 Catabolic gene alkM-F 5′-CGGCTACTCCGATGATCGG-3′ 62 870 3 Table 1. Oligonucleotide primers used to identified hydrocarbon degrading bacteria alkM-R 5′-GATCCGAGTGCCGCTGAAG-3′ 62 3 ndoM-F 5′-CACTCATGATAGCCTGATTCC-3′ 62 642 3 ndoM-R 5′-CACAACACACCCATGCCGCTG-3′ 62 3 todM-F 5′-GGGCTTACGACACCGCCG-3′ 62 560 3 todM-R 5′-GCGCTCCACGCTACCCAG-3′ 62 3 xylM-F 5′-CTGCGTGTACTGGACATGAG-3′ 62 834 3 xylM-R 5′-CGGTGGTCCAGGTCACCG-3′ 62 3 1—Arellano and Olmos, 2002; 2—Personal communication (Sandra Nierzwicki Bauer); Olmos, 2003; 3—The present work, primers modified from Whyte et al. (1996). Oligos for Hydrocarbon Degraders

  6. Detection by PCR Amplification Lane 1: Molecular Weight Marker Lanes 2-8: PCR products from DNA extracted for soil suspensions using Pseudomonas primers Lanes 9-15: Bacillus primers

  7. DNA Hybridization

  8. DNA Sequencing

  9. TRFLP

  10. Real-time TRFLP Yu, et al. AEM 71:1433-1444

  11. DGGE

  12. Genome Sequencing

  13. Microarrays

  14. TaqMan Real-time PCR Images by Dan Pierce

  15. Image by Dan Pierce http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Pierce/realtimepcr.htm

  16. Other Techniques • Stable Isotope Probing (SIP) • Fluorescent In Situ Hybridization (FISH) • mRNA Analysis

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