Topic 2 - Isolation of nucleic acids and proteins Flashcards
promoter def
cis element upstream of a gene where proteins initiate gene transcription
mRNA def
spliced transcript; many genes are spliced more than one way
ORF def
open reading frame; starts with start codon, ends at stop codon
(describes mRNA instead of using “exons”)
5’ UTR and 3’ UTR def?
untranslated regions
- in genomic DNA, pre-mRNA, and mRNA
where is polyA addition signal?
genomic DNA, pre-mRNA, and mRNA in 3’UTR
intron is where?
genomic DNA and pre-mRNA
NOT mRNA
exon is where?
genomic DNA and pre-mRNA
called ORF in mRNA
sense/antisense strand in transcription context?
antisense strand is transcribed to make pre-mRNA, not the same for every gene
sense strand = same sequence as pre-mRNA
what can go wrong under improper sample storage conditions?
- degradation, community change, temp/light/oxygen/pressure change, contamination, dynamic transcription
what should we aim for in sample storage?
- use fresh material if possible
- if not, use material that has been frozen QUICKLY
– ice crystals can penetrate into cells otherwise - liquid nitrogen or dry ice should be used for flash freezing -> no ice crystal penetration
what can we do in the field without access to freeze (sample storage)?
can use products like DNA/RNA shield (zymo) or RNA later (invitrogen)
- lyse cells and inactive nucleases and infectious agents (e.g., viruses)
general steps for isolation of nucleic acids (and proteins) (5)
- step 1: collect sample with nucleic acid of interest
- step 2: homogenize/lyse tissue and/or cells
- step 3: remove large cellular debris
- step 4: remove small non-target molecules (purify nucleic acid)
- step 5: concentration (and further cleaning) of nucleic acids
different methods of mechanical/physical lysis
- sonication
- high pressure/shearing
- mortar and pestle
- freeze thaw
- bead beating
different methods of chemical lysis
- changing pH
- detergents (e.g., SDS)
methods of enzymatic lysis
e.g., lysozyme
often cells/tissues are added to buffer first, before lysing.
common buffer components (4) and their function?
EDTA (ethylene diamine tetraacetic acid)
- chelates (grabs) Mg2+ (essential cofactor for nucleases)
SDS (sodium dodecyl sulfate)
- detergent that disrupts cell membranes
Salts (e.g., Tris-HCl, NaCl)
- regulates pH and osmolarity (Tris-HCl)
- aids in protein removal (NaCl)
how do detergents lyse cells?
e.g., SDS has hydrophobic tail and hydrophobic head (like in phospholipid bilayer).
When added, it makes “mixed micelle,” disrupting the membrane
what is a lysate? what could it contain?
liquid containing components of lysed cells
- DNA, RNA, proteins, cell membranes, organelle components, etc.
during step 3: remove large cellular debris, what happens?
centrifuge
will also remove other large debris if present (e.g., soil)
DNA/RNA are in solution (supernatant here), small compared to everything else
during step 4: Remove small non-target molecules (purify nucleic acid)
what do we use? what precipitates in what order?
Remove RNA from DNA extraction
- Use RNase (very heat stable, work carefully with it)
Remove DNA from RNA extraction
- DNase
Protein removal
- Protease treatment
- Phenol chloroform extraction
during phenol chloroform, what precipitates?
- Depending on sample matric, may have other compounds to precipitate out of DNA solution (e.g., humic acids removed from DNA extracted from soils)
- Generally precipitated, and centrifugation is used to remove them
from bottom to top:
- phenol layer (w/ lipids)
- protein precipitate (protein)
- aqueous layer (DNA and RNA)
is phenol more or less polar than water?
has a benzene! less polar :)
what are 2 options for step 5:
concentration and further cleaning of nucleic acids?
option 1: ethanol/isopropanol precipitation
option 2: spin column
ethanol/isopropanol precipitation: what happens? notes?
add ethanol and salt solution -> DNA/RNA precipitates -> centrifuge and remove liquid -> DNA/RNA pellet -> wash with 70% ethanol to remove salt in pellet
- fragments <100bp do not precipitate effectively
- precipitation removes residual phenol if present
- can be used after nucleic acid extraction to concentrate and re-suspend nucleic acids in a new solution (Useful when your protocol involves multiple steps with diff enzymes)
- salt neutralizes charge, DNA more likely to compact
spin column: what happens?
- under high salt conditions, DNA/RNA preferentially binds to the silica membrane (-ve charged) within spin column
- inorganic and organic materials will not bind
- centrifuging spin columns (or applying a vacuum) passe the liquid through spin column. DNA/RNA stays bound, everything else passes through
- before eluting DNA/RNA, a “wash buffer” is passed through column to help wash away unbound stuff
- water or a low-salt buffer is passed through column -> no longer high salt conditions -> DNA/RNA unbinds from membrane, passes through with elution buffer
spin column QUICK steps (4)
- add high salt conc buffer to DNA
- bind DNA to membrane
- wash
- elute
If you forget to mix your DNA solution with salt before applying it to the spin column, which of the following would be true?
a) The DNA will still bind to the silica membrane as DNA has a high affinity for silica.
b) The DNA will pass through the silica membrane during the initial centrifugation step.
c) You can still add the salt after you centrifuge, it isn’t too late!
d) You will likely end up with an undetectable amount of DNA at the end of your extraction.
b) and d)
while using micropipettes, nucleic acids are sheared into ____ kb fragments?
when does this matter?
- 30-50 kb fragments
- matters during genome sequencing
controls for DNA extraction:
- you get a good yield. how do you know this is from your sample?
- you get no yield. how do you know your sample contained no detectable DNA?
- negative control
- positive control
Ex. You’re extracting RNA from lead tissues of 10 different species of plant to sequence the mRNA and look for differences in genes being transcribed in each of the 10 plant species. In this situation, technical replicates would be…
a) Three leaves taken from one individual plant.
b) One leaf from each of the ten plant species.
c) One leaf, homogenized and split into three subsamples.
c)
Ex. You’re extracting RNA from lead tissues of 10 different species of plant to sequence the mRNA and look for differences in genes being transcribed in each of the 10 plant species. In this situation, biological replicates would be…
a) Three leaves taken from one individual plant.
b) One leaf from each of the ten plant species.
c) One leaf, homogenized and split into three subsamples.
a)
when would we need single cell DNA/RNA extraction? (2 common scenarios)
- sample contains very few or just one cell
- cells with high level of genomic variation, could mask effects of interest
(Cell heterogeneity is important in normal development (e.g., brain development, aging) and development of disease (e.g., cancer). Seeing the heterogeneity on a cell by cell basis rather than mixing all cells could be important for studying these biological phenomena.)
single cell DNA extraction:
often end goal -> sequence extracted DNA/RNA
what do we NEED for sequencing, in this case?
what is important??
- requires a whole genome amplification step to have enough for sequencing
- important to have highest possible DNA quality
single cell DNA extraction:
why is high DNA quality so important? how do we ensure this?
- loss of quality in one DNA segment in one cell cannot be compensated by another
- maximize quality by: not bead beating, not harsh lysis method, be careful with pipetting (shearing)
Summary for DNA or RNA extraction:
(5 steps & key considerations, single cell)
- collect sample
- think about sample storage - lyse cells
- select a lysis method - remove large cellular debris
- remove small non-target molecules (DNA, RNA, proteins)
- DNase, RNase, protease, or phenol chloroform extraction - concentrate/clean nucleic acid of interest
- ethanol/isopropanol precipitation (phenol chloroform) or spin column (DNase, RNase, protease)
single cell extraction:
- need high quality DNA
- need whole genome amplification before downstream analyses
how big are plasmids without an insert?
3-5 kb (Small!)
characteristics of plasmids (5)
- small (3-5 kb without an insert)
- supercoiled
- circular, ds
- 10^3x smaller than EC genome
- common in prok, sometimes found in euk
isolation of plasmid DNA:
genomic DNA, plasmid, method name for isolation of plasmids from genomic DNA
- during cell lysis, the genomic DNA is broken into linear, non-supercoiled fragments
- plasmids stay supercoiled bc of small size
– during plasmid isolation, handle gentle to keep plasmid DNA supercoiled; avoid nicks - alkaline lysis!!!!
isolation of plasmid DNA:
obtaining a sample steps (4)
- grow o/n culture containing plasmids
- add antibiotic (select for cells w/ plasmids) - collect cells via centrifugation
- resuspend cell pellet in buffer
- lyse cell
- gentle! try not to shear genomic DNA or damage plasmids
isolation of plasmid DNA:
alkaline lysis method
pH of solution w/ DNA is raised to 12!! (denaturation)
- disrupts cell membrane, lyses cells
- denatures DNA
- interacts with hydrogen atoms so cannot H bond
- genomic DNA no longer supercoiled
pH suddenly neutralized (renaturation)
- genomic DNA aggregates and precipitates because nothing is holding them together
- plasmid DNA just re-H bonds (DNA strands are still interlocked)
(centrifuge)
isolation of plasmid DNA:
purifying/concentrating the plasmid (method)
- can use a spin column like genomic DNA/RNA extraction
– in presence of high salt conc, plasmid DNA binds to -ve charged beads; contaminants are washed away and plasmid DNA is eluted (cation bridge)
– can use vacuum or centrifugation, like for genomic DNA/RNA
isolation of plasmid DNA:
purifying/concentrating the plasmid -
(buffer, where is plasmid after alkaline lysis, how is it different from genomic DNA extraction)
cells are resuspended in an alkaline buffer with RNase -> lyses cells (without shearing dmg) and denatures genomic DNA
- solution is neutralized and genomic DNA precipitates
- centrifuge -> ALL non-plasmid cellular components in pellet, plasmids in the supernatant!
- buffer with high salt conc added, whole mixture added to silica spin column
- wash & elute like in DNA/RNA extraction
how is protein extraction similar to DNA/RNA extraction?
same general premise:
- lyse cells
- remove DNA and RNA, other cellular components
– DNase, RNase, spin column
- precipitate protein to concentrate
protein extraction:
what is a “purified” protein? why would we want this? what does this usually involve?
isolated protein from other proteins
often interested in a specific protein, not all proteins in a cell
most commonly involves column chromatography
explain gel filtration chromatography, AKA, how does it separate proteins?
separates proteins based on size
AKA size exclusion chromatography
- small and big proteins start at top of column
- add buffer to wash proteins through
- bigger proteins will elute first; small proteins get stuck in polymer bead pores
- collect fractions
how is gel filtration chromatography different from gel electrophoresis?
it’s kind of the opposite!
small proteins get stuck in the bead pores, so bigger proteins elute first (travel “farther”), unlike in gel electrophoresis
explain ion-exchange chromatography, how does it separate proteins?
separates proteins based on charge
NOT always +ve charged beads
- +ve & -ve charged proteins start at top
- if using +ve charged gel bead, -ve proteins get “stuck”, will elute slower
- collect fractions with salt solution:
salt interacts with column and outcompete proteins, due to higher amt of salt compared to proteins
protein purification:
how can we detect proteins in each column fraction?
- if protein of interest = enzyme, can test enzyme activity in each fraction
- protein fractions are often separated by protein gel electrophoresis options (not normal gel electrophoresis):
1. SDS PAGE
2. 2D PAGE
why can we not use normal gel electrophoresis for separating proteins?
proteins can be positive, negative, or neutral
(used for DNA bc DNA is all -ve charged)
(1D) SDS PAGE
(stands for, separates on basis of ____)
sodium dodecyl sulfate
polyacrylamide gel electrophoresis
- separates based on molecular weight (size)
- similar to agarose electrophoresis
2D PAGE
(separates on basis of _____)
separates proteins based on:
- first: isoelectric point
- second: molecular weight (size)
1D SDS PAGE steps
treat sample
- often involves boiling sample with:
– a reducing agent which disrupts disulfide bonds between cysteine residues (don’t want tertiary structures)
– SDS, coats proteins in an even -ve charge (all proteins run in same direction)
small molecules go through matrix faster
polyacrylamide provides a sieving effect, separating proteins based on molecular weight
proteins detected afterwards by staining (e.g., coomassie blue)
what does each band in SDS PAGE represent?
one molecular mass of protein in mixture
- can have multiple proteins of same size within any given band
limitations of SDS PAGE?
- each band may be a mixture of similar proteins of same molecular weight
- so many polypeptides in a crude extract -> impossible to detect a specific protein by staining alone usu.
- spectrum of proteins that are resolved depends on protein abundance
- do not see protein complexes or native protein composition (dimers, tetramers, etc.)
2D PAGE steps
- Separates first based on isoelectric point (first dimension), and then based on molecular weight (second dimension)
- ideally: find a spot for each polypeptide loaded
what is 2D PAGE used for, from a research perspective?
- detect members of a gene family or find evidence of post-translational modification
general steps of 2D PAGE + how is 2nd step diff from SDS PAGE?
step 1: separation based on charge (IPG strip)
- protein mix added to IPG (gel strip w/ pH gradient)
step 2: separation based on molecular weight
- like standard SDS page, key diffs:
– instead of adding each protein fraction to a well, load whole IPG strip on top of gel
– instead of boiling protein mix in reducing agent, treat IPG strip with SDS and reducing/alkylating buffers (buffer permanently disables tertiary structures)
what does IPG in IPG strip stand for?
immobilized pH gradient
how does the IPG work?
an electrical field is applied, and proteins migrate toward the opposite charge.
as they migrate further towards the opposite charge, the protein’s charge decreases as pH changes along the gel strip
when a protein reaches a NET NEUTRAL charge, it stops migrating. the pH at this point is the ISOELECTRIC POINT of the protein
2D PAGE: instead of boiling the protein mixture in a reducing agent, what do we do? why?
treat IPG strip with SDS and reducing/alkylating buffers
- buffer -> permanently disables tertiary structures
- purpose: SDS -> everything has same charge
- denature the protein, destroy sulfide bonds
- alkylating buffer “caps” cysteine residues to stop them from forming disulfide bonds
What does 2D PAGE look like compared to 1D (SDS PAGE)?
more “dot-like” rather than lines in 1D gels
more likely to be individual proteins because of charge and size separation
what do bigger dots mean in 2D PAGE?
more protein of that charge and size
+ve / -ve controls of 2D PAGE
positive control:
- sample known to contain protein of interest
- should have a band for protein of interest
negative control:
- sample known to not contain protein of interest
- should not have a band in the expected position of protein of interest
benefits of 2D PAGE
- can be used to compare proteomes of two cell populations; datasets can be shared
- spots can be cut out of gel for amino acid sequencing and identification
- allows distinguishing/detection of related gene family members & related forms of one polypeptide
limitations of 2D PAGE
- difficult to do for membrane proteins (same for SDS PAGE) (hydrophobic)
- proteome detected depends on extraction methods and gel conditions
- highly abundant proteins make large spots that can distort results/hide other spots
