🧬 DNA & RNA Fundamentals
What does it mean that DNA strands are antiparallel? which way does it run?
The two strands run in opposite directions: one runs 5′ → 3′ and the other runs 3′ → 5′.
What is the Central Dogma?
DNA → RNA → Protein
Why is proper handling especially important for RNA specimens?
RNA is susceptible to degradation, so appropriate handling and timely processing help preserve the RNA.
DNA has an overall (pos or neg charge?)
Negative charge
Overall purpose of PCR
To amplify/copy a specific target region of DNA.
What type of bonds hold complementary DNA bases together?
Hydrogen bonds
mRNA:
tRNA:
rRNA:
mRNA: Carries genetic information from DNA for protein synthesis.
tRNA: Carries amino acids and uses its anticodon to recognize complementary mRNA codons.
rRNA: Major structural and functional component of ribosomes.
What does the A260/A280 ratio tell us?
It helps assess nucleic acid purity. For DNA, a ratio around 1.8 generally indicates acceptable purity.
DNA migrates toward the
Positive electrode
PCR cycle? Describe each step.
Step 1: Denaturation
Double-stranded DNA separates into single strands.
Step 2: Annealing
Primers bind to complementary sequences on the DNA template.
Step 3: Extension
DNA polymerase extends from the primers and synthesizes new DNA.
Why can the two strands of DNA be separated without breaking the DNA backbone?
The hydrogen bonds between complementary bases are relatively weak compared with the covalent phosphodiester bonds of the DNA backbone.
What occurs during transcription?
DNA serves as a template for synthesis of RNA by RNA polymerase.
What must happen to cells before DNA can be isolated?
Cell lysis
What allows the probe to bind its target?
Complementary base pairing/hybridization.
Why must PCR polymerase be thermostable?
PCR involves repeated exposure to high temperatures during denaturation, so the polymerase must remain functional after heating.
Complete the complementary DNA strand:
5′ – G C T A A C – 3′
3′ – C G A T T G – 5′
Exon vs. intron
Exons are retained in mature RNA and contain sequences that can contribute to the final product. Introns are removed during RNA processing.
Wavelengths used to evaluate DNA purity
260 nm and 280 nm
Purpose of hybridization
To allow complementary nucleic-acid sequences to bind, which can be used to identify/detect a specific sequence.
Four PCR components
Template DNA
Primers
Thermostable DNA polymerase/Taq
dNTPs
Buffer/Mg²⁺
How does GC content affect DNA melting temperature?
Higher GC content increases the melting temperature/stability of double-stranded DNA.
Alternative splicing:
Different combinations of exons can be joined together, allowing one gene to produce multiple RNA/protein products.
Why are nucleated cells important for genomic DNA extraction from blood?
Genomic DNA is located in the nucleus. White blood cells have nuclei, whereas mature red blood cells lack them.
What is a nucleic-acid probe?
A labeled nucleic-acid sequence designed to bind specifically to a complementary target sequence.
PCR Troubleshooting
Scenario: Patient specimen = amplification signal; negative control = amplification signal.
1. What should you suspect?
2. Why is the negative control important?
3.Can the patient result be confidently interpreted?
Scenario: Patient specimen = amplification signal; negative control = amplification signal.
1. What should you suspect? Contamination, particularly contamination of the negative control/reagents with target/template nucleic acid.
2.It helps detect contamination or unintended amplification. A properly functioning negative control should not demonstrate target amplification.
3. No. A positive negative control indicates a problem with the run, so the patient result cannot be confidently interpreted as valid without resolving the problem and appropriately repeating the testing.