Test 5 Flashcards
What is in situ hybridization?
Detects positions of target mRNA, non-destructive.
What is northern blotting?
Measures size of mRNA after gel electrophoresis, destructive.
What is RNA sequencing?
A method of analyzing large sets of RNAs that involves making cDNAs and sequencing them, destructive.
What is immunofluorescence?
Detects position of target proteins, non-destructive.
What is western blotting?
Measures size of proteins after gel electrophoresis, destructive.
What is proteomics?
Study of the structure and function of proteins in the human body (proteome), destructive.
What is primary (direct) immunofluorescence?
One antibody chemically linked to a fluorophore, then attached to epitope on antigen.
What is secondary (indirect) immunofluorescence?
Primary antibody binds to target protein (epitope), secondary antibody binds to fluorophore, then binds to primary.
Best molecules for gel electrophoresis?
Smaller, negatively-charged, and tightly condensed globular molecules migrate faster.
What does it mean if no signal is detected in northern blot?
Gene is not expressed OR RNA sample is degraded. Use control probes to detect other genes that would also be present.
What is quantitative PCR?
Measures mRNA level of the same gene between samples.
What do PCR reactions need?
- dsDNA template
- Mixture of all four dNTPs
- Stable DNA polymerase
- Two primers for the opposite strands
- Aqueous buffer solution
What happens in PCR amplification phase 1?
Sample heated and template denatures into single strands.
What happens in PCR amplification phase 2?
Temperature reduced and primer hybridize to their complementary sequence.
What happens in PCR amplification phase 3?
Temperature increased for DNA synthesis (longer template takes longer).
What are the 4 major transformations?
- PCR and cloning
- Recombinant DNA
- DNA sequence identification
- Genome targeting (CRISPR)
What is the right primer (reverse)?
Upper strand.
What is the left primer (forward)?
Lower strand.
What do restriction enzymes do?
Recognize a specific sequence of bases anywhere within the DNA.
Which direction does DNA move in gel electrophoresis?
Towards positive charge, as it is negatively charged.
What are germline mutations?
Source of all genetic variation, occurs in gametes and is transmitted to progeny.
What are somatic mutations?
Occurs in somatic cells, mutation inherited by daughter cells.
What are gene mutations?
Small alterations that affect a single gene or locus.
What are chromosome mutations?
Large alterations that affect chromosome structure or number.
Where do mutations occur?
Anywhere.
What are the consequences of mutations?
Either no functional consequences or can affect gene function/expression.
What are mutations in protein coding regions?
- Can affect amino acid sequence
- Substitutions: missense, nonsense or silent
- Insertion/deletion: frameshift or in-frame
What are mutations in noncoding regions?
Can affect rate of transcription or post-transcriptional processes.
What is a transition point substitution?
Pyrimidine to pyrimidine or purine to purine (C <–> T or A <–> G), more frequent.
What is a transversion point substitution?
Purine to pyrimidine or pyrimidine to purine (A <–> C or T; G <–> C or T).