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
What are the phylogenetic methods of sequence analysis?
- Parsimony
- Distance (neighbor-joining)
- Maximum likelihood
What is the parsimony phylogenetic method?
Fewest evolutionary steps (base changes) to generate observed sequence differences
What is the distance (neighbor-joining) phylogenetic method?
Fraction of sites that vary between sequences
What is the maximum likelihood phylogenetic method?
Maximizes the probability of observing the sequence data
What is bootstrapping?
Statistical procedure that resamples a single data set over and over again to create simulated samples
What is the difference between genomics and metagenomics?
- Genomics: single organism
- Metagenomics: many organisms simultaneously
What are some NextGen sequencing methods?
- Illumina: shorter reads
- PACBio: longer reads
What is a gene probe?
Typically a fluorescent-labeled single stranded sequence of DNA or RNA that is used to search for its complementary sequence in a sample genome
- Often used to find functional genes of interest
One way gene probes can be applied is in fluorescent in-situ hybridization (FISH). What is FISH?
Lab technique used to detect and locate a specific DNA sequence on a chromosome
How is FISH done?
- Cells are preserved whole
- Cells are permeabilized with chemicals
- Cells are hybridized with labeled probes that bind to the cell’s DNA or RNA
- Upon excitation, the fluorescent probes emit light
What is real-time/qPCR?
Technique that allows us to follow in real time the amplification of a target gene
- Can be done with DNA or RNA
To do a qPCR with RNA, what must the RNA first be converted to? How is this done?
cDNA
What are the caveats/limitations of qPCR?
- Still PCR based (see earlier issues)
- Control DNA/RNA reactions must be used
- 16S rRNA gene numbers do not equal cell numbers
Imagine you are a scientist conducting research on microbial genetic material. Give an overview of the methods you would use/what you would do.
- Nucleic acid extraction
- Run a PCR
- Clone the PCR product
- Extract the DNA from that
- Sequence the DNA (can do several things with the DNA sequence)
- Run the sample through BLAST
- Analyze the sequence (phylogeny)
- Gene probes
