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).
What is a missense mutation?
A mutation that changes one amino acid.
What is a nonsense mutation?
Changes a normal codon into a stop codon.
What is a silent mutation?
A mutation that changes a single nucleotide, but does not change the amino acid created.
What is a conservative mutation?
The new amino acid is of the same type as the original (chemical properties are similar), usually no effect on protein function.
What is a nonconservative mutation?
Chemical properties of mutant amino acid are different from original amino acid, effects protein function.
What is a frame-shift mutation?
Insertion or deletion of a nucleotide in the DNA sequence.
What is an in-frame mutation?
Insertion or deletion of a trinucleotide set that does not change the reading frame of the codon.
What are loss of function mutations?
Reduced/abolished gene activity, recessive inheritance.
What is a null mutation?
Complete loss of function.
What is a hypomorphic mutation?
Reduced gene function.
What is haploinsufficiency?
When one gene copy is not sufficient for organism to survive.
What is a gain of function mutation?
A mutation that confers new or enhanced activity on a protein, dominant.
What is a hypermorphic mutation?
Generates more gene product or the same amount of a more efficient gene product.
What are neomorphic mutations?
Generate gene product with new function or that is expressed at at the wrong time or place.
What is a forward mutation?
Wild type to mutant form (A to a), more frequent.
What is a reverse mutation?
Mutant to wild type (a to A).
What is CRISPR/Cas9?
Bacteria protects bacterial genomes from invading DNAs.
What is adaptation CRISPR?
Cas complex integrates small pieces of viral DNA into CRISPR array of host cell.
What is expression CRISPR?
CRISPR array transcribed into long pre-crRNA and processed by Cas proteins. crRNA will partially base pair with tracrRNA and form effector complex with Cas.
What is interference CRISPR?
Needs PAM; complementary base-pairing between crRNAs and viral DNA; Cas protein cleaves viral DNA.
What do the effector complex and PAM do?
Unwind nearby DNA.
What is the DNA endonuclease?
Cas9.
What is the protospacer adjacent motif?
NGG sequence in DNA, not on sgRNA.
What is nonhomologous end joining?
Error-prone DNA repair pathway for DSBs, creates random indels.
What is homology-directed repair?
Repairs DSBs by replacing damaged DNA with another DNA molecule by homologous recombination.
What is Duchenne muscular dystrophy?
X-linked recessive; usually in boys.
What is a model organism?
Easy to raise, reproduce in lab, rapid life cycle, numerous offspring, inbred populations.
What is convergent evolution?
Different organisms independently evolve similar traits.
What is forward genetics?
Driven by phenotype to genotype, identify the gene mutation that causes the mutant phenotype, germline.
How to do forward genetics?
Linkage mapping with positional cloning or DNA sequencing.
What is reverse genetics?
Genotype to phenotype, disrupt the gene sequence to identify functions.
How to do reverse genetics?
Knockouts, RNAi, CRISPR.
What are genetic screens for forward genetics?
Gain or loss of function mutations, in vivo mutagenesis, loss of biochemical pathways (microbes).
What is knockdown?
synthesized siRNAs silence gene expression, temporary effect.
siRNAs are complementary to mRNA of gene
What is a disadvantage of knockdown?
Can’t explain which gene is underlying the phenotype.
What is a knockout?
Removing/inactivating a gene from the gene sequence
What is a conventional knockout?
Target gene is knocked out in all tissues at all times.
What is a conditional knockout?
A gene that can be selectively deactivated in specific tissues when this happens.
What are global knockout limitations?
Hard if gene is in multiple places, deleting a gene in one place can affect it in another, hard to determine cause of phenotype.
What happens when both loxP are on the top strand in the same direction?
DNA between them is removed.
What happens when one loxP is on the top and one on the bottom in opposite direction?
DNA between will be inverted.
What are transgenes?
A gene from one organism is put into another at random locations.
What is the goal of transgenes?
Gain info about function of gene products and identify regulatory sequences.
What are transgene limitations?
Variability, random insertions, codon bias, post-translational mods.
What is whole genome shotgun sequencing?
Randomly breaking up genome into small DNA fragments, sequence individually, then reassemble.
What is annotation?
Identify the location of genes and functional sequence in genome.
