Biological Lab Techniques

freeze drying

proteins can be stored by freezing

reduce pressure to trigger sublimation to remove water

heating proteins will denature them

ELISA

enzyme-linked immuno-sorbent assay

looks for specific antibodies or antigens

ex: used to screen for anti-HIV antibodies

method:

1. wells coated with specific antibody/antigen

2. sample added, antibodies bind to antigen

3. everything else washed away

4. detection enzyme-linked antibody added

5. solution added that changes color if enzyme present

RIA

radioimmunoassay

looks for specific antigens

similar to ELISA, but uses radiolabelled antibodies rather than enzyme-linked antibodies

ex: used to measure amount of hormones

method:

1. radiolabelled antigen is mixed with antibody

2. radioactivity is measured

3. unlabelled antigen (cold) is added to compete off the radiolabelled antigen (hot) to make a standard curve

4. last step is repeated with sample instead of unlabelled antigen to measure amount of antigen

gel electrophoresis

separate nucleic acids or proteins by size or charge

negatively charged samples migrate towards positive terminal (anode), small things migrate faster

method (for DNA):

1. add restriction enzymes to cut up DNA (at palindromes)

2. add loading dye to samples

3. run electrical current

4. compare distance sample migrated to ladder

SDS-PAGE

denatures the proteins and masks the native charge so that comparison of size is more accurate, but the functional protein cannot be recaptured from the gel

reducing conditions break disulfide bonds and break up dimers/trimers

separates by molar mass, smaller things migrate faster through the gel matrix

a protein with excessive positive charge may travel a shorter distance that expected

southern blotting

looks for specific DNA sequences

ex: used to monitor mutations

method:

1. add restriction enzymes (they like palindromes)

2. run DNA gel electrophoresis

3. transfer to nitrocellulose paper

4. ss hybridization probes added, complementary to target

5. probes have radiolabeled nucleotides for visualizing

northern blotting

southern blotting except with RNA

western blotting

looks for specific proteins

native gel- run without denaturing/reducing to retain protein structures

1. lysate is denatured, SDS to make negatively charged

2. run gel electrophoresis

3. transfer to nitrocellulose paper

4. primary antibody added

5. secondary antibody added, enzyme-linked to fluoresce

eastern blotting

looks at post-translational modification of peptides

isoelectric focusing

separate proteins based on pI

set up pH gradient in gel, then run electrophoresis

proteins move to the pH that equals their pI (where they have 0 net charge, and are not attracted to electrode)

2D gel can be run to separate by pI and molecular weight on two different axis

recombinant DNA

DNA is transcribed/translated in a different organism

restriction endonucleases- bacterial enzymes that cut specific sequences of DNA, creates sticky ends

restriction sites are usually palindromes

DNA fragment can be inserted in a plasmid that was also treated with the same restriction enzyme

plasmids

circular ds-DNA in bacteria with autonomous replication

elements:

origin of replication- determines number of copies

cloning site- for restriction enzymes

resistance genes- for plasmid selection

uses:

1. transformation- plasmid enters bacteria, uses host machinery for transcription/translation, can integrate too!

2. cDNA- introns are a problem, so use reverse transcriptase to generate cDNA from mRNA then insert

3. artificial chromosomes- bigger, better than plasmids

4. transfection- eukaryotic plasmids into mammal cells

PCR

polymerase chain reaction

amplifies and looks for specific DNA sequences

ex: screening for hereditary diseases

method:

1. DNA, primers, nucleotide bases, DNA polymerase added

2. denaturation- sample heated, open DNA strands

3. annealing- sample cooled, primers attach

4. extension- sample heated, replication

5. repeat for 30 cycles

6. visualize with electrophoresis

primers should have high GC content, especially at the two ends, to ensure strong binding

RT-PCR

reverse transcriptase-polymerase chain reaction

looks for specific mRNA sequences

method:

1. all mRNA isolated and converted to cDNA with reverse transcriptase

2. PCR

3. visualize with electrophoresis

qPCR

quantitative or real-time polymerase chain reaction

looks for amount of DNA sequence, gene expression

DNA amount measured each cycle with fluorescent probe

DNA sequencing

determine entire genome

DNA sequence can be read from bottom of gel to top, after extrapolating the sequence of the template strand

ex: constructing primers

Sanger technique:

1. dNTPs need OH on C3 to link together

2. modified dNTPs that lack C3 OH to terminate elongation

3. denature DNA into single strands

4. radiolabeled primers to show you where it starts

5. mix with dNTPs and some modified dNTPs of A only that randomly insert to generate a variety of fragments

6. repeat with C, G, T modified dNTPs

7. visualize with electrophoresis

genomic sequencing

genetic linkage map- hundreds of markers per chromosome, create BAC libraries with chromosomal fragments, fragments are separately sequenced

shotgun approach- chromosomes cut into fragments, cloned, sequenced, use computer to align fragments

goals:

1. where genes are located, organized

2. genetic variation across species

3. cancer genome

DNA fingerprinting

restriction fragment length polymorphism (RFLP) analysis:

1. polymorphisms- highly variable DNA

2. restriction enzymes to cut up polymorphic DNA

3. separated with gel electrophoresis

4. southern blotting to look for specific sequences

short tandem repeat (STR) analysis:

1. PCR to amplify small stretches of polymorphisms

2. gel electrophoresis, south blotting

other methods for studying genome

exome sequencing- target certain regions for sequencing

karyotyping- staining whole chromosomes for major genetic changes, aneuploidy, some insertions/deletions

fluorescence in situ hybridization (FISH)- use fluorescent probes to find specific DNA sequences on chromosomes, used to study translocations

microarrays

looking at relative RNA amounts

ex: comparing transcriptional profile of two people

method:

1. RNA samples are mixed with a fluorescent dye

2. applied to an array chip that has binding sites for many different genes

3. lights up as dye color if that gene is present

4. quantify based on color intensity

ISH

in situ hybridization

looking at location of gene expression in tissue sample

method:

1. tissue sample is fixed to slide

2. labelled probe is added for DNA/RNA of interest

3. enzyme-linked antibody is added

IHC

immunohistochemistry

looking at location of protein in tissue sample

ex: stain for estrogen receptor to help breast cancer pts

1. primary antibody specific for target protein is added

2. secondary antibody is added

flow cytometry

looking at number of cells expressing markers

ex: separate T cell populations using CD4/CD8 markers

method:

1. cells are stained with antibodies linked to fluorescent tag

2. cells passed through beam of light one at a time

3. light scattering and light emission is measured

4. fluorescence-activated cell sorting (FACS)- sort cells based on expression of markers

protein quantification

use different techniques to quantify protein over time, location, in different environments, drugs, etc.

Bradford quantification- Coomassie blue stains proteins, UV-vis spectrophotometer to determine protein quantity