biotechniques and experiments needed Flashcards
Griffith experiment (1928)
AIM: observe and prove the transformation principle
2 strains of strep used:
1. R strain: non virulent
2. S strain: virulent due to polysaccharide capsule that protects them from immune system
EXPERIMENT: creation of 4 mice
1.R strain mice lived
2.S strain mice died
3. heat killed S strain mice lived
4. !!! mix of heat killed S strain and live R strain mice DIED
CONCLUSION: transformation principle - a molecule/some material was exchanged between killed S and live R cells that transformed R cells and made them virulent. ALSO TRANSFORMATION IS HEREDITARY
Avery experiment (1944)
AIM: identify whether RNA/DNA/proteins was the transforming factor
EXPERIMENT:
-removal of lipids and carbs from sample so only DNA/RNA/proteins were present
-preparation of 4 tubes using S strain cell extract:
1. control tube containing all 3
2. tube with protease (no proteins) –> only DNA/RNA
3. tube with DNAse –> only RNA/proteins
4. tube with RNAse –> only DNA/proteins
!!introduction of R strain bacteria in each of the tubes
CONCLUSION: DNA is the transformation principle and carrier of cell genetic material because transformation only failed to occur in the tube containing DNAses
Hershey and Chase experiment (1952)
AIM: use of bacteriophages to prove whether proteins/DNA are inserted into cells in transformation (further proof of what was shown by avery)
EXPERIMENT: two sets of bacteriophages cultivated:
1. in radioactive sulfur medium
2. in radioactive phosphorus medium
-infection with E coli
-separation by centrifugation
CONCLUSION:
-Ecoli injected with S35 phages were not radioactive meaning the proteins were not incorporated in bacterium
-Ecoli injected with P32 phased WER radioactive meaning DNA was incorporated into cell
HENCE: DNA is the transformation principle
CHARGAFF experiment and conclusion
-used TLC to separate the bases of DNA
CONCLUSIONS: (4)
1. 4 nucleotides are not present in equal amounts
2. different species have different nucleotide content
3. A=T and C=G
4. cells making up tissues within organism share the same nucleotide content
diameter of DNA
2nm
!!!! stays constant because a purine is always being paired with a pyrimidine and so their combined length doesn’t change
how many base pairs can fit in one period of DNA
10.5
(period = same point going down helix)
why is RNA a lot more unstable and reaction
OH on carbon 2 of the ribose sugar opens itself to attack
Fast vs slow renaturation of DNA
FAST: rapid cooling of denatured DNA –> lower probability of collision between complementary bases on strands –> increases hybrids that contain irregular base pairings –> LIMITED RENATURATION
SLOW: gradual temperature cooling of DNA –> higher probability of correct collisions so produces highly/fully renatured strands
Relationship between extent of DNA denaturation/renaturation and absorption using photospectrometry
Absorption increases as denaturation increases (due to destruction of nitrogenous rings present in double helix which exposes the bases more)
HENCE:
single stranded DNA has highest absorption, fast renatured DNA has mid absorption, slow renatured DNA has low absorption
spectrophotometry
used to determine concentration of DNA due to direct proportion
!!SOS: RNA has a higher absorbance than DNA because it is single stranded
Gel electrophoresis of DNA
-separation of DNA fragments based on their size/molecular weight
-uses agarose gel with electrolyte solution and an electrical supply
RNA electrophoresis
-requires denaturing gel (containing formaldehyde) to eliminate any secondary folding
CHARACTERISTICS OF A SAMPLE THAT IS INTACT:
1. 28S and 18S prominent bands of the rRNA (most abundant)
2. slight smear of mRNA between them
protein electrophoresis
-SDS PAGE: sodium dodecyl sulfate polyacrylamide gel
-proteins denatured and boiled in SDS, which breaks 3ary structure, prevents reformation of higher structure and coats with negative charges
! reducing agent added for breaking of covalent bonds like S-S
-molecular weight in daltons
restriction enzyme use
-hydrolysis of phosphodiester bonds in specific location without the use of ATP
-sticky ends created
Southern blot
identifies a specific gene within a mix of DNA post gel electrophoresis
-use of a nylon filter
-use of a radioactive probe
(not used much now because PCR and sequencing are easier)
how are the results of a northern blot interpreted and verified
housekeeping gene is used to see if the same amount of RNA has been added into the different wells
Northern blot
determines extent of gene expression via detection of specific RNA molecules in sample of RNA
-use of nitrocellulose filter
-use of radioactive probe
FISH
located genes present on chromosomes and can identify chromosomal translocation
In situ hybridisation
localises specific nucleic acid targets to determine gene loci
-fixation of tissue using paraffins and addition of probe
PCR
Amplifies DNA sample of interest
-addition of: sample DNA, dNTPs, primers, TaqP
-3 rounds of temp changes
-denaturation, annealing, extension
rtPCR
Amplifies RNA targets by converting it into cDNA
-incubation with reverse transcriptase and oligo primers for cDNA production
-PCR is then run
!! SOS: determination of target sequence is QUANTITATIVE
microarrays
-detects expression of several genes simultaneously (TRANSCRIPTOME OF CELL- all RNA transcript sequences)
-RNA isolation and reverse transcription into dCNA
-labeled with diff fluorescent markers and placed in oligo-containing wells
Sanger sequencing
-sequences exact base sequence of a gene
-separation into 4 vessels to add each ddNTP (halts synthesis)
-separation using electrophoresis
-visualised with autoradiography
automated equencing
-more efficient version of sanger sequencing
-fluorescent labeling of dNTPs and detection via computer
pyrosequencing
-very fast sequence of bases
-relies on the release on pyrophosphates during DNA rep
monoclonal antibodies vs polyclonal antibodies
MONO: recognizes only a single epitope of an antigen and is extremely specific
POLY: produced by multiple immune cells and so have the affinity for the same antigen but different epiptomes
EPIPTOME: specific part of an antigen recognised by an antibody
monoclonal antibody production
-identify the presence of a protein by being antigen specific
-antigen injected into animal which produces antibodies for it
-antibodies hybridised with myeloma cancer cells for fast replication
Western blotting
Identify a specific protein within a protein extract separated by SDS PAGE using antibodies
-incubations (2) with antibodies that are labeled with fluorochromes
-housekeeping genes are also used for regulation
ELISA test
-enzyme linked immunosorbent assay
2 types: antigenic and serological
-informs of the presence and abundance of an antigen
-serum added to wells containing fluorescently marked antibodies and intensity of fluorescence observed shows qualitative abundance of antigen present
!FASTER version of western blot
immunofluorescence microscopy
-detection and localisation of antigens within tissue slice
-tissue extracted and embedded on slide
-fluorochrome marker
-visualised under microscope
