Molecular Analysis (3 blots & other techniques): detecting DNA, RNA, pros

difference between 3 blots (snow drop)

grind up tissue to get DNA, RNA, pro

• southern blot: DNA fragment identification

• northern blot: same but with RNA or mRNA

• western blot: a protein identified

southern blot (DNA fragment detection)

• gel is run to get different size fragments

• transferring fragments onto a membrane, a labeled (radioactive or fluorescent) probe of nucleotides (complementary to the target sequence) is added to the membrane, probe binds to the specific DNA fragment.

  • note: alkaline solution is needed to get ssDNA

northern blot (RNA or mRNA)

• RNA transcripts of different lengths

• Probe is labeled complementary nucleotides (same process of DNA southern blot)

  • note: no digestion cause already single stranded

western blot (different size pros- shape is important!)

• purpose: tells if specific pro is present, how big it is, and how much is present in a sample

  • SDS-page separates proteins by size

  • transfer pros to membrane (blotting part)

  • Probe is labeled antibody that bind to pro of interest

what method is used to see gene expression patterns?

Northern- RNA analyzed: RNA expression changes depending on the tissues (DNA is same in all cells)

FISH

works like northern blots to study gene expression patterns in tissues/whole organisms, helps to see where on a chromosome a gene is located

SDS-page, why used for western blots?

a special type of gel electrophoresis

• pros have different charges, shape, and sizes

• SDS denatures the pro (all linear), coats them with a negative charge! so protein- fragments move by size

aspects of cell identity:

• genome: DNA

• transcriptome: all RNA made

• proteome: all pros made

different cell types: same genome, diff transcriptome & proteome

what are housekeeping genes?

genes that are always on bc needed for cell to function

(ex. actin genes, are controls in experiments)

other analysis techniques: what do scientists sometimes to with RNA

convert it to cDNA & this represents only expressed genes

other analysis techniques: RT-PCR (rev transcriptase PCR)

alternative method to northern blotting for RNA observation

• use RT to convert RNA to cDNA (expressed gene)

• do PCR on it to amplify that gene

so if PCR product is detected, gene was being expressed

other analysis techniques: qRT-PCR (qualitative RT-PCR)

purpose: find how much RNA is present (how much expression)

if more starting RNA, there will be more PCR product

materials for SDS page

• SDS (detergent) coats pros in - charge

• heat: denatures proteins so all linear

• BME: breaks disulfide bonds

other methods for RNA analysis:

• RNAse protection assay

• RNA sequencing

• microarray

other protein analysis method

immunolocalization

how does RNAse protection assay work?

a super sensitive method, where a target RNA strand & labeled RNA probe bind

• RNAse enzyme eat up any ss RNA that didn’t bind

• analyze protected fragments on gel

what is RNA sequencing method for RNA analysis?

purpose: looking at multiple genes at same time

RNA is broken into small fragments, turned into cDNA

cDNA is fed into sequencer

therefore, all mRNA is analyzed

what is a microarray (RNA) for analysis? did in MI!!

purpose: looking at many genes at same time

• specific DNA probes are attached to a (chip)

• Sample RNA is labeled with a fluorescent dye and can bind the probes

• A scanner detects the brightness of each spot, correlating to the amount of RNA present

what is immunolocalization for protein molecular analysis?

purpose: find exactly where a protein is, in a cell or tissue

• antibodies are probes: primary antibody binds to pro of interest

• 2ndary antibody (has a dye marker) binds to primary antibody

• sample has parts that will then glow- can look at it under microscope