Chapter 10 Flashcards
Purpose of detecting and quantifying DNA,RNA, and proteins
- Investigate their function in normal and diseased states
- Determine if certain genes are present in similar species
Purpose of blotting
Detect SPECIFIC molecules within a mix of many different molecules (DNA, RNA, protein)
Steps of Blotting
- Gel electrophoresis
- Transfer molecules to membrane paper
- incubate membrane with probe solution (hybridization)
- Autoradiography (X ray)
In Situ Hybridization (ISH)
In vivo version of blotting for RNA and DNA
Radioactive probes
fluorescence in situ hybridization
uses fluorescently labeled probes instead of radioactive probes
Immunofluroescence
In vivo version of blotting for proteins
uses antibody probes and a fluorescence microscope
Traits of restriction enzymes and sites
- Endonucleases
- Cleave phosphodiester bonds
- sites are palindromic (both strands have same sequence in antiparallel fashion)
PCR purpose
Make billions of copies of fragment of dna
PCR steps
- Pair of primers (20 nuc each) designed to base pair to end of target gene
- Added to solution with template, dATP/dGTP etc. and Taq
- Heated to denature, cooled to aneal, heated to synthesize
Real Time PCR
Purpose: calculate amount of DNA in a sample at a given time
Measure intensity of fluorescent signal generated by dye
Calculation: X=2^(CT1-CT2)=2nd sample has X fold less DNA than 1st sample
where CT is the cycle threshold (number of cycles for signal to be detected above ground)
Used to compare relative ampunt sof diff DNA in sample
Example of purpose of qPCR
1.diagnose cancer due to a somatic mutation - determine fraction of cells in tumour sample that contain mutant gene vs WT
- compare relative amount of same dna ( rate of progression of viral infection in blood samples taken at different times)
PCR for RNA
- Purify mRNA from tissue or specific cells
- Incubate with reverse transciptase, dNTPs and oligo-dT primner (anneals to polyA tail)
- Convert to cDNA (double stranded version of mRNA molecule)
Use cDNA as you would DNA in same tests
multiple cloning site/polylinker
Restriction enzyme site that does not repeat in a plasmid - plasmid only cuts at one location
Plasmid Vectors
- Plasmid Vectors: replicated independently of whole chromosome due to ori site.
- Good for drug resistance testing and determine which bacteria contain resistant genomes
PRACTICAL: dna inserts disrupt gene lacZ in plasmid. it encodes enzyme B-galactosidase - cleaves compound X-gal when added to culture plate and turns blue.
THEREFORE: with insert - white. without - blue
SMALL INSERTS
Expression Plasmids
- contain sequence that controls transcription and translation of insert dna
- can drive transcription constitutively (constant) or inducible (when signaled)
PRACTICAL:
1. pET plasmid 1 have T7 promoter - drives transcription of inserted gene in E.coli - expresses phage T7 RNA polymerase
- pET plasmid 2 uses lac operon components to inducible express gene in e.coli - lac operator site near T7 promter, contains lac1 gene - encodes lac repressor protein - binds lac operator - represses transcription. When IPTG added to growth media, lac repressor inactivated and T7 transcribes
- pET have epitope tag (His-tag). Epitope tags : purify recombinant proteins. short protein sequences - translated in frame at N or C terminus of recomb protein. Purified by affinity chromatography from E.coli extract - solution of e.coli cells broken open release recomb protein and e.coli protein - mixed with charged beads - only recomb bind to beads - tags bind to beads with recomb - beads washed - only recomb beads left - released by adding competing chemical. USED TO SYNTHESIZE AND PURIFY HUMAN INSULIN PROTEIN
BACTERIOPHAGE VECTORS
- can hold different sized of inserts (double stranded)
- central part of genome removed with RE and replaced with insert
- Up to 15kb
- can be introduced to cells via phage
MEDIUM SIZE INSERTS
Vectors for large inserts
- Fosmids
- Bacterial artifical chromosomes (BACs)
Fosmids
- 35-45 kb inserts
-fosmid packaged into A phage particles - particles introduce insert into recipient e.coli cells
- cos sites (cohesive) : 12 bp sticky ends - turn linear phage dna into circular
- fosmids then replicate extrachromosomally (similar to plasmids)
- few copies made per cell (normally one)
BACs
- 100 to 200 kb
- dna inserted
- plasmid introduced to bacterium