Molecular Genetic Techniques 2 (L6) Flashcards
cDNA library screening goal
for obtaining a full-length coding sequence of the target gene for subsequent characterization
cDNA library screening steps
- cells from each colony on nitrocellulose filter
- incubate filter w/ alkaline solution to lyse cells, denature DNA
- hybridize w/ labeled probe
- wash away labeled DNA that does not hybridize to DNA bound to filter
- autoradiography
signal appears over plasmid DNA that is complementary to probe
plasmid
circular, extrachromosomal DNA that can replicate independently inside bacteria
DNA cloning steps
- Restriction enzyme - sticky ends on DNA vector
- Restriction enzyme to cut plasmid.
- Enzymatically insert DNA into plasmid.
- Transform plasmid into bacteria.
- Antibiotic resistance selection - only transformed cells survive.
- Create large colony of bacteria w/ copies of DNA you want to amplify.
what can be done to plasmids/what do they have that allows for DNA cloning?
- polylinker region/ multiple cloning site where you can engineer a series of restriction enzymes sites
- origin of replication - can replicate inside bacteria
- ampicillin resistant gene
reverse transcription mechanism
- Hybridize mRNA w/ oligo-dT primer (recognize polyA tail)
- Transcribe RNA into cDNA.
- Remove RNA w/ alkaline conditions.
- Add polyG tail at 3’ end of ss cDNA w/ terminal transferase
- Hybridize w/ oligo-dC primer.
- Synthesize complementary strand w/ DNA pol.
- End w/ ds cDNA sequence
how can you protect cDNA?
methylation at EcoR1 sites
Once you have ds cDNA, what can you do with a plasmid?
Transform cDNA into a plasmid, then use same DNA cloning procedure to create more full length sequences
how can a probe be synthesized from a plasmid?
add primer and nt’s to create a restriction enzyme site so that restriction enzyme sites are automatically incorporated into amplified DNA - then make probe by using DNA pol to transcribe ssDNA w/ sequence from target region
fragile X syndrome diagnosis
done with southern blot
FMR1
fragile x mental retardation 1 - gene that causes the disease
- get > 200 CGG or CpG island repeats in promoter
- when tri-nt are expanded, triggers methylation of CpG islands and of repeats -> gene repressed -> problems
northern blot
used to compare mRNA expression in different conditions
-slight difference from southern blot: can also evaluate level of expression (thickness, darkness of band)
what do microarrays tell you?
know which gene is expressing at what level in your sample
genomics
study of whole genome of an organism
microarray colors
green: decreased expression
red: increased expression
heatmap
tells expression of genes in different patients - important concept: using gene profiles as a better means of studying diseases
what is the technique for protein characterization?
SDS-PAGE = sodium dodecyl sulfate - polyacrylamide gel electrophoresis
what is the purpose of SDS?
detergent - denatures protein and adds an even coating of negative charge on the polypeptide
why do you need a reducing agent for protein characterization?
to break disulfide bonds to get single polypeptide chains coated with SDS
what are two reducing agents?
mercaptoethanol and dithiothreitol
ratio of SDS to aa’s and implications
1 SDS/ 2 aa’s - nice even coating
SDS-PAGE steps
- Denature with SDS.
- Place mixture of proteins on gel and apply electric field (proteins separated by size).
- Stain to visualize separated bands (Coomassie blue).
western blot
- SDS-PAGE
- Transfer to membrane.
- Incubate with primary antibody (binds target protein).
- Wash.
- Incubate with enzyme-linked secondary antibody (binds primary antibody)
- Wash.
- React w/ substrate for enzyme on secondary antibody.
- Visualize.
what is the purpose of using two rounds of antibodies in western blot?
signal amplification
clinical application of western blot
research and diagnosis (HIV)
protein separation w/ 2D gel
- separate proteins by isoelectric focusing
2. separate proteins by size (SDS-PAGE)
isoelectric focusing
create a pH gradient, then apply electric field -> proteins migrate to where they are neutral
isoelectric point
point where polypeptide has no charge
advantage of 2D gel separation?
gives finer map of protein distribution
3 types of column chromatography
- gel filtration
- ion exchange
- antibody affinity
basic concept of column chromatography
use a polymer matrix to separate proteins based on different properties
gel filtration chromatography
smaller proteins get stuck in beads, large ones cannot -> larger proteins move through column faster, come out first
ion exchange chromatography
polymer has charge that will bind proteins of opposite charge - ones w/ same charge come out first, then elute other proteins bound to column
antibody affinity chromatography
only target proteins bind to antibody on the beads of the column -> gives you a concentrated, purified fraction
proteomics
large scale analysis of proteins
mass spectroscopy
used to identify and sequence proteins