Study Questions Set 5 Flashcards
Distinguish between the terms “mutation”, “DNA repair” and “recombination”.
- mutation transmissible permanent change in nucleotide sequence of a chromosome
- DNA repair mechanisms by which mistakes in DNA sequence are corrected
- recombination exchange of genetic material between two DNA molecules
Explain how errors in DNA replication can lead to mutations.
- if the error is not corrected, the sequence changed can have an effect
o regulatory sequences might cause changes in regulation/transcription
o exons could change protein product - if mismatches are not corrected, half the daughter chromosomes of meiosis contain the wrong base
List the 3 major causes of mutation in DNA
replication errors
spontaneous change
external factors
Explain Southern blotting
Southern:
- DNA detection
- Uses of gel electrophoresis for the detection of a specific DNA sequences in a sample of DNA
- Denatured using alkaline solution
- bound with labelled probe, viewed by X-ray radiography
Explain Northern blotting
- RNA detection
- Geared towards detecting gene expression
- Technique is similar to Southern blotting but no denaturion is needed
Compare the information obtained by northern analysis with the information obtained by microarray experiments.
- Northern analysis only obtains one gene for each probe
* Microarrays obtain multiple genes – can study complex interaction between genes simultaneously
What kind of information can we obtain from a northern blot?
• From a northern blot you can obtain:
o The steady-state level of a specific transcript
o What transcripts are made at different conditions
o Determination of the gene that codes for the gene of interest
What kind of information can we obtain from a Southern blot?
• From a southern blot you can obtain:
o Detection of repetitive sequences
o The number and position of gene copies
o Detection of transgenes
How do we treat a DNA gel prior to Southern blotting. Explain why.
• DNA is denatured with an alkaline solution to separate the double stranded DNA into single-stranded DNA. Only ssDNA can be bound by the probe.
Thinking question: you have made a short probe (50 nucleotides) from a certain genomic DNA (note: genomic DNA includes both exons and introns). Are you sure that you would be able to use this probe for northern hybridization? Explain your reasoning.
• No, northern hybridization detects mRNA
o mRNA are intron-free, they only have exons
• The probe from the DNA contains both introns and exons
• If the probe has introns, it will not be complementary to the mRNA inefficient
• Since the probe is short, the chances of finding its complementary base pair and sticking to them is unlikely
• This is dependent on it being eukaryotic; if prokaryotic, it is likely an acceptable probe
What is a template?
• Template: a strand of DNA or RNA, used in genetic sequencing to synthesize new strands (i.e. complementary strand for mRNA synthesis)
What is a primer?
Primer: a nucleic acid strand that serves as a starting point for DNA replication. The primer provides a 3’ OH group for DNA polymerase to add appropriate nucleotides for polymerization.
What is a probe?
• Probe: a nucleic acid used for the detection of complementary nucleic acid sequences. The probe will hybridize with the target to be detected. It can be genes from DNA that code for a specific protein, or it can be used to identify mRNA.
o Man-made DNA sequence (small) detects target gene
Why is it important to know the exact start site of transcription?
- The exact start site of transcription is important because it is from there that the promoter region is defined.
- DNA regulatory sequences (controls transcription) are often near the start site of transcription.
- Transcription and translation starts sites are different!
What does SDS-PAGE stand for? Explain the roles of SDS in SDS-PAGE (keep in mind - two major roles)
• SDS-PAGE stands for sodium dodecyl sulphate polyacrylamide gel electrophoresis.
• SDS is negatively charged detergent
o Binds to hydrophobic protein regions – helps protein unfolding
o Applies a negative charge to each protein in proportion to its mass.
Without SDS, different proteins with similar molecular weights would migrate differently due to differences in folding. Differences in folding patterns would cause some proteins to better fit through the gel matrix than others. SDS linearizes the proteins so that they may be separated strictly by molecular weight regardless of intrinsic charge
