-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”

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 fractionation Size 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