09b: Modern Techniques Flashcards
To isolate genomic DNA for analysis, which cell(s) are used for sample?
Lymphocytes
To isolate mRNA for analysis, which cell(s) are used for sample?
The target tissue
To isolate proteins for analysis, which cell(s) are used for sample?
The target tissue
Genomic DNA is different in which specific tissue type(s)?
SAME IN ALL CELLS
Genomic DNA is used for analysis to investigate:
- Genetic mutations
- Gene promoter sequences
- Exon/intron junctions
Genomic DNA cannot provide information about:
Any tissue-specific expression
T/F: Fully processed mRNA is the only type of mRNA that can be isolated and analyzed.
True
mRNA is used for analysis to investigate:
- Tissue-specific gene expression levels
- Expression level differences in normal and tumor cells
- Different developmental stages
- Transcriptional/post-transcriptional modification
mRNA cannot provide information about:
Gene structure
Proteins are used for analysis to investigate:
Post-translational processing (phosphorylation, proteolytic cleavage, etc)
All sequences cleaved by restriction enzymes are:
DNA palindromes
(X) are bacterial enzymes that have evolved as protection mechanism (protect from invading organisms).
X = restriction enzymes
Restriction enzymes can generate which types of ends?
- Blunt
2. Sticky (3’ or 5’ overhangs)
(Blunt/sticky) ends are more precise.
Sticky
In Southern blot, the blot contains (X) and the probe is (Y).
X = DNA Y = DNA
In Northern blot, the blot contains (X) and the probe is (Y)
X = RNA Y = DNA
In Western blot, the blot contains (X) and the probe is (Y).
X = protein Y = antibody
In (X) blots, the probe is (single/double)-stranded DNA of (known/unknown) sequence.
X = Southern and northern
single-stranded, known sequence
List ways in which a Northern blot probe can be generated.
ssDNA probe can be generated via:
- Synthesis
- Restriction fragment
- PCR
Couple wants to know if they’re carriers of sickle cell mutation. You take a sample from which tissue(s)?
Lymphocytes (to isolate genomic DNA)
How does the probe stick to the sample (DNA/RNA/protein) on a Southern blot?
DNA; the DNA probe is ssDNA and the DNA on the blot is denatured, ssDNA as well. Complementary binding of probe to target sequence
You’re using (X) blot to check for specific disease mutation. Which sequence do you put on the probe?
X = Southern
ssDNA containing the mutation
In blot, which tool is used to see the potential adherence of probe to sample?
Probe is labeled with radioactive (32)P before incubated with blot; then expose to X-RAY
What’s RFLP? What is it used for?
Restriction Fragment Length Polymorphism; used to identify mutations which affect restriction sites
The (X) mutation in sickle cell disease impacts (Y) site. Thus, a normal sequence will differ in mutated sequence in which way?
X = point (Glu to Val) Y = restriction
Normal sequence will be cleaved, but SS sequence will not be cleaved
How does the probe differ in RFLP and in southern blot?
It doesn’t differ
Could you tell a SS carrier apart from SS homozygous patient via RFLP?
Yes - one allele differs between them, so the carrier would have one mutated and one normal (total of 3 bands instead of one on gel)
The incredible thing about PCR is:
Ability to replicate DNA in quick cycle, due to heat-stable Taq Polymerase
How is PCR used in forensics?
Make primers for the universal “flanking regions” of STR sites
How does CRISPR work?
- Virus invades bacteria
- “Spacer” sequence derived from virus and integrated into CRISPR sequence
- CRISPR RNA foremed
- CRISPR RNA guides Cas9 nuclease to target and destroy viral genome
What’s cDNA?
Copy of DNA that’s complementary to specific RNA sequence
(X) can be used to produce cDNA from (Y).
X = reverse transcriptase Y = RNA sample
