Nucleic Acid Measurements

In: Science

Submitted By 8AM25PM
Words 1309
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Lecture 8
Nucleic Acid-Based Measurements Text Chapter 13

Wednesday, July 24, 2013

Total community DNA
• Extract DNA from soil
– – – – remove cells from soil separate cells from soil lyse cells separate DNA from cells – purify DNA

• Extract DNA from soil
– Extract DNA from cells in presence of soil
• Bead-beating • chemical or enzymatic treatment
– Sodium dodecyl sulfate or lysozyme

Wednesday, July 24, 2013

DNA purification
• Cesium chloride gradient centrifugation • Kits

Low density DNA

High density

Wednesday, July 24, 2013

Visualizing nucleic acidsBlotting
• Southern blotting
– DNA

• Northern blotting
– RNA

Wednesday, July 24, 2013

Agarose gel electrophoresis
-

Stain gel with ethidium bromide +
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DNA purification
Agarose gel verification

Wednesday, July 24, 2013

Gene Probes
• Phylogenetic probes
– 16S rRNA

• Functional gene probes
– dsr (bisulfite reductase) sulfate reduction – nirS (nitrate reductase) nitrate reduction

Wednesday, July 24, 2013

16S rRNA gene probes
• Oligonucleotide primers for PCR
Target region

cDNA

16S rDNA clone library

• Oligonucleotide probes complementary to 16S rRNA molecule
– no need for PCR because many copies in cells
Wednesday, July 24, 2013

cDNA RNA ribosome

Secondary Structure: 16S rRNA
Different locations on the 16S rRNA molecule offer identity at different phylogenetic levels •Domain EU338 •Phylum •Class •Family •Group CF319a •Genus •Species •subspecies
Wednesday, July 24, 2013

Fluorescent in situ hybridization (FISH)

Wednesday, July 24, 2013

Protein synthesis

From: Curtis & Barnes, Biology, Pg 315.

Wednesday, July 24, 2013

Hybridization Assay fixation/permeabilization fixative substratum cells hybridization oligo probe washing

visualization
Wednesday, July 24, 2013

mounting…...

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