biotechnology and microscopy Flashcards
(29 cards)
Types of microscopy:
Optical (aka light) or Electron
Types of optical microscopy, advantages and disadvantages?
Compound microscopy:
- visible light focused on a thin sample, producing 2D image. Can see many cells, tissues, and organisms.
- Disadvantage: Samples thicker than one cell layer require staining (kills specimen).
Fluorescence Microscopy:
– fluorescent probes used to tag structures. Can see where specific molecules (e.g. proteins) are located in the cell.
- Advantage: Fluorescent markers do not kill sample, allowing live viewing.
Types of Electron Microscopy, advantages and disadvantages
Scanning Electron Microscopy (SEM):
– electron beams shot across surface of sample, producing highresolution 3D images of surface (texture, shape). Visualizes the exterior of very small objects (e.g. ribosome).
- Disadvantage: Sample must be dehydrated and coated before viewing (kills specimen).
- Memory Tip: SEM –> “S” for Surface
Transmission Electron Microscopy (TEM):
- electron beams shot through a thin section of sample, producing high-resolution 2D images. Visualizes internal structures of tissues, cells, and organelles.
- Disadvantage: Samples must be dehydrated, fixed, and sliced (kills specimen).
- Highest magnification microscope.
What is the highest magnification microscope?
Transmission Electron Microscopy (TEM)
Relative size and density of cellular components (which one comes out to the pellet layer first)
Nuclei > Mitochondria/Chloroplast > Microsomes > Ribosomes/Viruses
(largest/most dense comes out to the pellet layers first and we can take it out
Types of Horizontal Gene Transfer:
Horizontal gene transfer – gene transfer between different, unrelated organisms. Can occur naturally or via biotechnology.
Conjugation:
– DNA transferred directly from one cell to another via a pilus (bacterial appendage used to exchange cytoplasm and small molecules).
Transduction:
- DNA transferred from cell to cell by a virus.
Transformation:
– DNA taken up by cell from environment.
-To transform a cell, it must first be made competent to promote spontaneous DNA uptake via heat-shock or electroporation (processes that increase membrane permeability).
How can you make recombinant DNA?
Recombinant DNA – DNA which contains segments from different sources. DNA segments can be exchanged via horizontal gene transfer or using artificial recombinant technology.
Can make it either through HGT or Artificial Recombinant Technology
Recombinant DNA technology:
Use restriction enzymes to cut DNA at sequencespecific, palindromic “recognition sites”.
- Sequences cut with the same restriction enzyme can bind together.
- Palindromic sequences: nucleotide sequences that read the same in the 5’ –> 3’ direction on both strands.
After new DNA sequence is introduced, DNA ligase seals the strands together, creating recombinant DNA
Restriction Fragment Length Polymorphisms (RFLPs), applications?
Different individuals will have differing nucleotide sequences, containing recognition sites at different locations in their DNA. Digestion with restriction enzymes produces unique DNA fragment patterns and lengths for each individual.
Application: DNA Fingerprinting – use RFLPs to match an individual’s DNA to a sample (e.g. crime scene).
Current DNA sequencing method vs early method
Current (next-gen sequencing):
Early method (sanger sequencing, aka dideoxy chain termination):
- uses primers, DNA polymerase, dNTPs, fluorescently labeled ddNTPs, and gel electrophoresis to decipher an unknown DNA sequence.
* During normal DNA elongation, new phosphodiester bonds form between the 5’ phosphate of a new nucleotide and the 3’ OH of the previous nucleotide.
* ddNTPs lack a 3’ OH, preventing DNA elongation –> If ddNTP is incorporated, DNA elongation terminates prematurely.
* In Sanger sequencing, ddNTPs get incorporated randomly, resulting in DNA fragments of varying lengths, each containing a fluorescently labeled terminal nucleotide.
-Each fluorescently labeled ddNTP (A/G/T/C) radiates a different color.
*DNA fragments are separated by size using capillary gel electrophoresis and a sensor detects the nucleotide-specific fluorescent color of each fragment size to interpret the DNA sequence.
What is cDNA, what is it used for, and how is it made?
Reverse Transcription: reverse transcriptase creates cDNA (complimentary DNA) from an RNA template.
RNA template has no introns. and therefore cDNA has NO INTRONS bc its created off of mRNA
* Retroviruses replicate using reverse transcriptase
Artificial eukaryotic cDNA is inserted into prokaryotic cells to mass-produce proteins.
* It is advantageous to express cDNA in prokaryotes rather than regular DNA in eukaryotes because prokaryotes are not capable of alternative splicing, which can alter gene expression.
* cDNA is preferred over mRNA since mRNA is unstable and short-lived
how much increase in DNA molecules in PCR?
1 –> 4 –> 8 –> 16 –> 32 –> 64
(2^n increase, where n = number of cycles)
DNA Microarray Assay and the steps
Used to view expression of multiple genes simultaneously.
- load cDNA into microwells and see if they hybridize
- allows us to see which genes are turned on/off in diff cells (because of fluorescent labels)
Steps to Microarray:
1. A microarray chip’s wells are loaded with ssDNA fragments, each fragment corresponding to a gene.
2. mRNA from a cell is reverse transcribed into fluorescently labeled cDNA.
3. Newly synthesized cDNA is added to each well.
4. If hybridization occurs, the probe fluoresces, indicating active gene expression.
Types of blotting, what is each for:
Southern blotting
Northern blotting
Western blotting
Southern blotting – detects DNA fragment of interest.
Northern blotting – detects RNA fragment of interest.
Western blotting – detects protein of interest.
SNOW DROP
S - southern D - DNA
N - northern R - RNA
O
W - western P - Protein
Steps to Southern Blotting (analogous to N and W):
- Extract DNA with gene of interest and digest using restriction enzymes.
- Separate DNA fragments by size (gel electrophoresis).
- Transfer fragments to nitrocellulose filter paper. (suction effect pulls DNA onto nitrocellulose paper)
- Expose nitrocellulose paper to labeled DNA probes.
- nitrocellulose paper is porous paper that allows DNA probes to bind better, also more endurant to washing & labelling - Visualize DNA fragments for presence of desired segment.
Immunofluorescence staining, advantages?
Visualizes where a specific protein is expressed in cell.
- Addition of primary antibody – binds to protein of interest.
- Direct immunofluorescence – Primary antibody contains a fluorescent tag. - Addition of fluorescently-tagged secondary antibody – binds to primary antibody.
- Indirect immunofluorescence – Secondary antibody contains a fluorescent tag. - Visualization – secondary antibody fluoresces –> visualizes where protein is expressed.
- Advantage: Can be performed in live cells.
Transgenic Animals
have genes introduced from the genome of a different individual (often different species).
What is a genomic library?
“backup copy of an organism’s genome”
- collection of cloned DNA pieces from a genome
- can be screened to locate a gene of interest
How do you make a genomic library?
Genomic Library – collection of cloned DNA fragments which represent full genome of an organism.
Steps to creating a genomic library:
1. Extract genome from organism of interest.
- Digest genome using restriction enzymes.
- these cut at specific recognition sites, leaves sticky ends - Cut a plasmid (containing antibiotic resistance genes) with same restriction enzymes.
- also will have sticky ends - Ligate fragments into plasmid to form recombinant DNA.
- seal using DNA ligase, now you have a recombinant plasmid DNA - Insert recombinant DNA plasmid (vector) into bacteria (via transformation - HGT).
- Vector: anything used to transfer foreign DNA into another cell. - Bacteria multiply and replicate their inserted plasmids, thereby replicating the genome.
- DNA can be re-isolated from the bacterial culture when needed.
Antibiotic resistance test: isolates bacteria that contain the recombinant genetic material. Only recombinant bacteria will contain the antibiotic resistance gene and survive in the presence of antibiotics.
How can we prepare bacteria to take up a plasmid?
Via
- heat shock + CaCl2 (increases membrane permeability)
or
- electroporation (creates temporary pores in plasma membrane)
What is reproductive cloning and what are the steps?
Producing a new organism which is genetically identical to the ‘parent’ organism (donor of the nucleus).
- Isolate nucleated donor cell from parent organism (somatic cell).
- Isolate unfertilized enucleated (nucleus removed) egg from a separate organism (egg donor).
- provides place for clone to initially develop in - Electroporate enucleated egg to fuse with parent nucleus, forming an embryo.
- cell division occurs - Transfer embryo into surrogate mother.
- Surrogate delivers baby clone.
- The clone is genetically identical to the donor of the nucleus.
- Dolly the sheep was the first reproductively cloned mammal, proving that the nucleus from specialized cells can be used to create an entire organism.
‣ Evidencethat differentiated cells still retain the full genome that can give rise to a fully functional organism.
What did Pasteur’s Swan Neck Flask Experiment show?
Disproved the theory of spontaneous generation –> New organisms can only arise from existing organisms.
- Spontaneous generation: A sterile (devoid of microbes), closed system will spontaneously generate new life forms over time.
- Pasteur boiled a broth solution in a curved neck flask to kill all microbes, before letting the flask sit.
- Before the curved neck was removed, no microbes grew.
- After the curved neck was removed, microbes grew.
- Conclusion: Microbes did not spontaneously generate, they entered the solution from the surrounding environment.
Griffith’s Experiment
Genetic traits can be transferred between different bacterial strains via an unknown heritable substance. Coined the term “transformation”.
- Mice that were infected with solution of live R-strain bacteria and heat-killed S-strain bacteria died.
- Rough (R) strain: no protective capsule, nonvirulent (non-pathogenic because immune system can destroy it) –> Live infection is nonlethal by itself.
- Smooth (S) strain: protective capsule enhances virulence (pathogenic, immune system cannot destroy it) –> Live infection is lethal,
- but can also be heat killed, therefore non-virulent if S bacteria is heat killed by itself
- BUT heat killed S bacteria can transfer the protective capsule to living R bacteria (which then become virulent)
- Conclusion: The R-strain gained the ability to create a protective capsule via transformation of the heat-killed S-strain’s genetic information, conferring virulence.
Avery-MacLeod-McCarty Experiment
DNA is the heritable substance responsible for bacterial transformation.
- Similar to Griffith’s experiment, except different digestive enzymes (e.g. proteinase, DNase, RNase, lipase) were added to the solution before infection.
- Only DNase prevented transformation (infected mice survived).
- Conclusion: DNA must be the genetic information that transforms R-strain bacteria, allowing them to create a protective capsule
