Module 1 biotech

DNA cloning (3)

• Insert DNA sequence into a vector, and introduce to a cell

• cleave DNA target with restriction endonucleases

• join fragment with DNA ligase to a vector also cleaved with the same endonuclease = hybrid molecule

DNA probes (4)

ssDNA labelled with radioitopes or other labels

• probes complimentary sequence to target DNA

• hybridizing of labeled DNA probe to target les us identify sequence of interest among many others

• ss oligonucleotide probes can be synthesized from being complimentary to target DNA

Blotting

transferring macromolecules to solid membranou s support

ASO probing (2)

DNA detect single nt differences (mutations or polymorphisms)

• allele-specific probe only binds if the sequence matches

Sotuern blot

• detect DNA presence and rleative size

northern blot

detect RNA (usually mRNA) presence and relative size

• also reflects gene expression

PCR (3)

• mutation detection

• amplify low abundance DNA

• forensic analysis

CRISPR Cas9

• edit genomic DNA in a live thing

• Uses guide RNA to guide Cas9 to target sequence to cut it and edit the DNA

qPCR

• measures Gene expression

• cDNA used, from mRNA

• fluoresence is proportional to amount of amplified DNA

Northern Blot

• measures RNA presence, relative size

cDNA Microarray

• mRNA converted to cDNA

• measure expression of many genes at the same time

• color, and also intensity of fluoresence = expression

SDS page (3)

• protein separation by size

• unfolds AND gives them a uniform negative charge

• smaler proteins move farther

Western Blot

• uses antibodies

• gives presence and relative size of protein after SDS page after it migrates on gel

ELISA

• quantify how much proteins and detect based on antibodies

DIrect:: antigen coats well, test if sampe has antibodies against it

Sandwich: the well is coated with antibody, patient sample into well, then add another antibody that recognizes a diff part of the protein sample