-The methods section of the course covers chapters 21 and 22, not chapters 20 and 21 -Paper discussion on Tuesday - assignment due at the start of class

DNA Methods - Electrophoresis - Digestion - Hybridization - Cloning - Sequencing Protein Methods - Purification - Electrophoresis - Sequencing - Immuno precipitation

Gel electrophoresis separates DNA molecules by size by exploiting the negative charge of DNA -Commonly use agarose gel -Use a polyacrylamide gel for fine resolution -Use pulsed field gel for very large (>30kb) fragments

Restriction endonucleases cut DNA at specific sequences EcoRI -Isolated from bacteria -Cut short palindromes -Used to fragment DNA

DNA hybridization exploits the complimentary strand binding property of DNA -Use a “probe” - a labeled complimentary strand -A southern blot can determine the relative amount of DNA in a sample based on intensity of probe binding -A northern blot uses the same principle but with RNA -Micro array uses this principle in reverse: Known gene sequences (probes) are fixed to a plate, mRNA (cDNA) added, amount bound is detected

In situ hybridization - apply “probe” to detect gene expression in tissue - where is a gene expressed?

Gene cloning - isolation and amplification of DNA -Isolate fragment of DNA with gene of interest via a restriction digest -Incorporate into a “vector” -Vector must have 1) restriction sites 2) ability to replicate 3) a selectable feature -Bacterial plasmids have these properties

DNA library: clone multiple fragments of DNA into vectors -Can incorporate entire genome into a vector (genome library) -Can make DNA from mRNA templates via reverse transcriptase enzyme, make cDNA library of only coding regions -Can screen vectors via hybridization

DNA can be amplified in vitro by PCR -DNA fragment with region of interest is denatured -Primers on either side of sequence are added -DNA polymerase builds complimentary strand starting at primer (both strands) -Heat to denature and repeat cycle -Can rapidly amplify specific sequences of DNA

DNA sequence is determined by PCR with a “chain terminating nucleotide”

- Addition of dd nucleotides (Guanine in this example) will lead to sequences that stop at a G

Protein purification exploits unique properties of proteins Charge fractionationSize fractionation

Proteins can be separated by size if treated first Immuno blot (western) is similar to a southern or a northern blot, but visualizes protein with an antibody

Protein sequencing via Edman degradation -Chemical modification of a terminal amino group allows a single a.a. to be cleaved at a time -Passing the released amino acid through an HPLC column reveals its identity

Protein sequencing via mass spectrometry -Digest protein into fragments -Pass through a vacuum -Peptide fragment movement is determined by mass and charge -Sequence of fragments is analyzed to determine the amino acid sequence

Chromatin immuno precipitation (CHIP) determines if a protein is bound to DNA