Experiments + Procedures important in Molecular Biology Flashcards
PCR
Used to make multiple copies of a segment of DNA - amplifies the DNA
Gel Electrophoresis
use charges to move segments based on size (farther they travel, smaller they are)
can compare to known segments
X-ray crystallography
Used to visualize structure (or structural differences) of a molecule
Chromatin Immuno Precipitation (CHIP)
This is how we detect/analyze modifications - how we determine specific interactions between DNA and a protein - what sequences are used to bind?
It allows us to localize any DNA binding proteins (includes modified histones) to the base pair level in the genome in a living cell
(to look at protein binding to nucleic acids in the cell)
- to do this need an antibody to your protein or modification of interst (specific - binds very specifically to a short aa sequence (antigen))
- once we have an antibody… take cells, add formaldehyde - “fixes” - covalently bonds proteins to DNA
- Next break open cells, break the DNA into shortish (200bp ish) pieces (can use high frequency sound waves (sonicate), put through needle, restriction enzyme) (can check length with elecrophoresis)
- add antibody to protein of interest - precipitate antibody complexes
- remove the protein (reove covalent bonds introduced by formaldehyde)
- look at DNA sequence somehow (can use PCR or CHipseq)
- graph number of sequences we get at each position
- (in chip seq, sequence whole genome, takes a couple of days)
https://www.youtube.com/watch?v=fQYJnDxZktM
Biochemistry (protein purification)
Isolate (purify) proteins and nucleic acids… study activity in vitro
Example: Purification of DNA rep proteins:
1. break cell open
2. centrifuge so things that are very heavy (nuclei) go to bottom
3. wash everything else away, pick up nucleus
4. break it open - lots of diff proteins (wanna know if theres a protein that can do DNA Rep)
5. Throw in test tube + things needed to make DNA… see if DNA is made… It is!
6. Based on some separation mechanisms (charge in this case) pass through column. Get what doesnt stick (neg charge) at end of column, then run a solition through to pick up everything that did stick (+ charge)
7. Run an assay on what did stick - do we get DNA? Yes
8. Run another column, base off something else (ex: size) , small things take longer - run an assay on each group that comes out to see if DNA is made
9. and on and on until protein is isolated or have very specific columns that only isolate enzyme you care about
to use for making antibodies, determining structure, or in vitro assays
(combination of biochem and genetics (either forward or reverse) is most powerful
Forward Genetics
Make random mutations in organism or cell then look for indibiduals altered in process - dont have to make an assumption about how things work
(combination of biochem and genetics (either forward or reverse) is most powerful
Chromatin Conformation Capture
Asks whether a particular sequence is near another in the nucleus (whether particular fragments interact) by:
- fixing proteins with formaldehyde
- break into small pieces
- circularize with DNA ligase
- analyze DNA sequence of little circles (either by PCR (have to have defined primers, so can only ask if 2 sequences are near enough to be ligated into 1 circle) or by whole genome sequencing)
Which sequences are together physically but not in genome?
Chromatin Conformation Capture 3C
Are two regions defined by PCR primers close in space?
One vs One
Uses a heat map: intensity of boc made by cross section reflects how often those 2 sequences are found together
darker - sequences found together a lot
lighter = sequences not found together a lot
3C and 4C are more localized - need two PCR primers… need to know what you are looking for
Chromatin Conformation Capture - Hi-C
all vs all
- paired end reads, from small ligated products
- so much data requires a lot of analysis
- In Hi-C, make circles and sequence them all
- often not used because too much info
Chromatin Conformation Capture - 4C
One vs all
- chromosome conformatio capture on chip
- asks what sequences are found close to a particular known sequence
- uses inverse PCR followed by hybridization to a “chip” or microarray
- Uses a volcano plot (another type of heat map)
- 4C uses sequencing from 1 PCR primer - asks what DNA sequences are close to the PCR primer? “viewpoint”
- *allows you to pick out things at a particular place (viewpoint) … any circle with a given sequence will be produced and all the other sequences in that circle
Reverse Genetics
Have an isolated gene (DNA seq) from a protein (“signal”) of interest, make specific mutations and see how process is affected
Have to make an assumption
If there is another protein that is really important, will not see anything about that
(combination of biochem and genetics (either forward or reverse) is most powerful
DNA Fiber Assay
to look at DNA rep
In vitro DNA synthesis
followed by gel electrophoresis
to look at DNA replication
if certain proteins are required for it
In vitro txn
followed by gel electrophoresis
to look at transcription
if certain proteins are required for it
also length of transcripts made - e.g.if looking for splicing proteins
qPCR
to detect/quantify amount of DNA or RNA
tracks target concentration as a function of PCR cycle number in order to derive a quantitative estimate of the initial template concentration in a sample.
need everything for replication to occur (dNTPS, sequence-specific primers, buffer, DNA pol, DNA)
Western Blot
to detect/quantify particular proteins in a sample
Generally, the thickness of a band is proportional to the relative amount of protein in that sample
DNase 1 footprinting
(and chemical footprinting)
to look at protein binding to DNA in vitro
hard to look at moving things (RNAP)
CryoEM
to determine structure or structural differences
