Lecture 8: Molecular Diversity Flashcards
What are the 4 steps of nucleic acid extraction?
- Cell lysis
- Chemical or mechanical - Clean-up
- Proteinase K, phenolchloroform, CsCl gradient - Get rid of contaminants and inhibitory substances
- Alcohol precipitation and resuspension of DNA/RNA
Explain how to do a PCR.
- Denaturation
- Annealing
- Extension
What are the caveats/limitations of using PCR amplicons to assay the environment?
- Extraction biases
- Incomplete lysis, degradation, etc. - PCR bias
- Limitations to amplification
- Primer bias (primers not universal) - PCR is not truly quantitative
- One must return to the environment to check
Which of the following are caveats/limitations of PCR-based molecular diversity approaches?
A. PCR is quantitative.
B. Not all cells are equally “extractable.”
C. Even “universal” PCR primers do not amplify all microorganisms 16S rRNA genes.
D. Both B & C
E. All the above
A. PCR is quantitative.
B. Not all cells are equally “extractable.”
C. Even “universal” PCR primers do not amplify all microorganisms 16S rRNA genes.
D. Both B & C
E. All the above
What is the common type of vector used for DNA cloning?
Plasmid
What are examples of mobile genetic elements in bacteria?
Explain why a plasmid is a functional cloning vector.
- Can be replicated independent of the bacterial genome
- Functional markers added to vectors allow you to insert things into the DNA
Why clone if you already have DNA?
- Improves the quality and length of the sequence read
- PCR introduces a lot of errors in the sequence –> cloning gives you cleaner identical copies of the DNA fragment of interest
What are the steps of cloning a PCR product?
- Separation mechanism for mixed environmental PCR products
- Ligation of PCR product into plasmid vector
- Transformation of competent E. coli cells
- Screening by plating on selective medium
What is the purpose of extracting DNA after you have cleaned and selected your colonies with your plasmid of interest?
To verify that it has the correct fragment of interest
How do you do a DNA extraction?
- Extract DNA from candidate clones
- Purify the DNA
- Analyze the DNA on an agarose gel
What is the main purpose of cloning environmental 16S rRNA genes?
A. This aids in identifying E. coli species.
B. This separates out the individual 16S genes.
C. This allows us to see a diverse set of sequences in a single clone.
D. This allows us to test for antibiotic sensitivity of bacteria.
E. All the above.
A. This aids in identifying E. coli species.
B. This separates out the individual 16S genes.
C. This allows us to see a diverse set of sequences in a single clone.
D. This allows us to test for antibiotic sensitivity of bacteria.
E. All the above.
Explain what happens in Sanger sequencing.
- Computer reads each band of the material through a capillary gel in order
- Uses fluorescence to identify the terminal dNTP (building blocks for your new DNA strand)
- Laser excites the fluorescent tag
- Computer detects what was added
- Incorporates the dNTPs by DNA polymerase in in vitro DNA replication
- As it adds those terminal ends, it can flesh out the full sequence of the gene of interest
What are the pros and cons of Sanger sequencing?
A. Pros
- Low error rate
- Can read 800 bp at a time
B. Cons
- Time consuming
- Expensive
- Low throughput
What does NCBI BLAST do?
- Generate alignments between your sequences
- Calculate statistical significance of those matches
