Lectures 1-2 Flashcards
Nucleic Acid Hybridization
- Annealing of one strand to its compliment
- Can be used to find complements in a complex mixture
- Efficiency of hybridization can show how different the two strands are from one another
Restriction Enzymes
In bacteria used to cut foreing DNA
These recognize palendrome
cut in squence specific manner–> this cut site can serve as a landmark
Gel Electrophoresis
Seperate by size
Smaller peices move farther
Negative phosphate backbone allows for movement by voltage
Sothern Blotting
Transfer from gel to paper
Dentature DNA with strng base
Probe paper for homology to sequence of interest
Cloning
Isolation and amplification of specific DNA fragment
Restriction enzyme–> ID and isolate fragment of interest –> ligate this into cloning vector –> transform into bacteria –> Amplify
Bacterial Plasmid
Small closed circle
Carry antibiotic resistance
Positive transformations are ID by plating
Bacteriaphage lambda
double stranded linear DNA
Non essential regions of the genome are removed and replaced by DNA fragments (they contain genes for amplifying genome, protein coat synthesis and packaging DNA)
Cosmids
Gitted version of phage
only keep area at end for replicating and packaging
this leaves more room for DNA
Bacterial Artificial Chromosome (BACs)
Yeast Artificial Chromosomes (YACs)
Most useful and stable way to maintain a large peice of DNA
Can contain large peices of DNA but often undergoes recombination
Genomic Libraries
isolated DNA of interest –> fragment it to controlled size –> random fragment ligation into vectors –> transformed into host –> replicated
cDNA Library
Contruct library from mRNA
RNA isolated –> enrich it with poly A tail –> RT –> RNA degraded –> dsDNA formed –> ligate into vector
Abundance of clone can show expression pattern in specific tissue
Screening Libraries
colonies lifted from petri dish to nitrocellulose paper
bacteria are lysed on paper then the DNA is denatured
DNA probes teh hybridize
RT-PCR
Uses flouresence to quantify products at each step of the reaction
the resulting curve of product v. time can be used to compare template abundance in different samples
Multi-plex PCR
A single sample is simultaneously analyzed with multiple sets of primers
Use PCR to diagnose for presence of pathogens
(HIV)
Allows for detection even before infection manifest when it can most effectivly be treated
HIV: RNA RT–> DNA –> PCR
Cloning by PCR
Isolate mRNA the amplify with PCR then clone with vectors
Northern Assay
Enrich RNA by adding poly A tail
Seperate sample by Gel Electro.
transfer to nitro paper
then view
This can tell if mRNA for a gene is present and the expression level
Protein Electrophoresis
Denatured: coat with neg charge so only seperating by size
Non-denatured: compare changes in charge between 2 proteins
Isoelectric focusing(IEF): seperate by charge only (pI)
2 dimensional: IEF then denatured
Using antibodies to detect proteins
primary v. secondary antibodies
Primary: binds protein
Secondary: binds primary but is covalently tagged
location and abundance can be measured
Western Assay
protein seperated by size with gel electro.
transfered to nitro paper
primary antibody
secondary antibody that is coupled to enzyme
incubate in a substrate for the enzyme
light is given off when enzyme is functioning
shows location and abundance of protein
Immuniflourescence microscopy
Primary then secondary antibody(flourescent tag)
microscopy
tells if protein is expresesed and if it is in the proper location
Micro- Arrays
used to simultaneously analyze the expression of thousands of genes
propes are short ssDNA complimentary to all predicted human mRNA
Micro-Array type 1
isolate mRNA
make flouresenct labelled copy of mRNA
hybridized labelled coppy to micro-array
read signal
measures relative expressionlevel in a single sample
Microarray type 2
RNA is isolated from refrence sample and labelled with color
RNA is isolated from sample and labelled with diff. color
they are simultaneously hybridized
ratio between two colors to compare relative expression
Cluster Analysis
often MicroArray is used to find genes that are similarly expressed
and/or whose expression changes in a similar manner under different conditions
often genes dound in the same cludter frequently function in the samepathways or cellular process
RNA Interference (RNAi)
mechanism used by cells to protect themselves against FNA viruses
Contruct gene that will express a self complimentary RNA
introduce it into the cell
This dsRNA is recognized by Dicer and helicase
dsRNA is cut into small pecies and then on of the strands is removed
this is used as a compliment to dins other mRNA in cell and destroy them
