Environmental biotechnology Flashcards
(14 cards)
define Metagenomics
the study of genomes of whole communities of microscopic life forms
eg. from a handful of soil
vs genomics = single organism
what are three key technologies relevant for metagenomics?
PCR
Microarrays
NGS (Next Generation Sequencing).
what is metagenomics useful for? (in terms of discovery)
finding novel antibiotics or enzymes
ie. finding new genes
what are techniques that can be used to enhance the starting material for metagenomics research?
Stable Isotope Probing (SIP), separates microbes that actively metabolise and incorporate a selected heavy isotope
BrdU enrichment - BrdU is a thymine analogue with a heavy Br atom. identifies proliferating cells (Br incorporated into DNA)
SSH - Suppressive Subtraction Hybridisation
Explain briefly how SSH (Suppressive Subtraction Hybridisation) works to enrich DNA of interest from environmental samples
used to identify unique / differently expressed genes in “tester”, mixed with a “driver”
denatured + hybridised, common sequences –> tester-driver hybrids, unique sequences remain single-stranded
ie. suppresses abundant sequences, preferentially amplifies rare sequences
define transcriptome
the complete set of RNA transcripts in a cell or community
(just as a genome is the complete set of DNA)
define meta-transcriptomics
the study of the collective transcriptome of a community
reveals which genes are being ACTIVELY TRANSCRIBED, “functional genomics”
name the three main branches of analysis methods for metagenomics or metatranscriptomics
- Sequence-based analysis
- Activity-based analysis
- Direct sequencing of entire library
Main types of sequence-based analysis
PCR (gene-specific, RT-PCR…)
Microarrays (to assess types and numbers of organisms)
FISH (fluorescent in-situ hybridisation)
Integron analysis (not in exam)
SIP (Stable Isotope Probing) is what type of analysis
activity based analysis (as opposed to sequence-based)
what do microarrays detect?
mRNA - this reflects what is ACTIVELY being expressed
what is a microarray?
a glass plate with thousands of DNA probes (short single-strands complementary to a specific gene) fixed to it
explain how microarrays work
mRNA is converted to cDNA and labelled with a fluorescent dye
cDNA is washed over a microarray, hybridises with matching DNA probes
intensity of fluorescent signals indicate how much of that mRNA was in the sample
What is RNA-Seq?
relies on next generation sequencing techniques
for transcriptomics
extract total cellular mRNA (converted to cDNA for ease) from a single cell and sequence, align with genome.
