bio research Flashcards
ELISA TECH is used to detect?
antigens (proteins and cytokines) and specific antibodies
if they are testing for an antigen, what do they use?
they coat the wells with antibody specific to the antigen . then they wash it off after binding occurs. then a second antibody is introduced and linked to an detection enzyme.
if they are testing for an antibody, what do they use?
antigen goes directly to the wells to detect the antibody and then it is ,mixed with enzyme linked antibodies
elisa immunoassays vs radioimmune assays
elisa will have a color change whena presence of the diesired is present and deals with enzyme linked antiobodies
RIA is commonly used more for detecting certain hormones in patients serum… deals with mixture of radiolabeled antigen and antibody to cause a radioactivity that is compared to the standard curve
electrophoresis
separates dna rna or proteins by size
gel can be agarose or acymilide
direction includes the neg going to the pos side
thicker the gel, the smaller the pores so the small things are able to travel faster than the big ones
what is blotting
once electrophoresis is complete, the desired dna rna or protein will be isolated and placed on a nitrocellulose membrane and begin probing the fragment or protein
what does snow drop stand for
diff blotting
SOUTH = DNA NORTH = RNA WEST = PROTEIN
recombinant protein
basically when u clones genes of dna from diff organisms and trsnacribe/ translate to make a combination of it which can t hen help to cure diseases or work as treatment
what is the significance of the plasmid?
u place the desired gene into the plasmid so that it can grow with the bacteria
although plasmids are good, prokaryotes lack introns so how can plasmids process the exact gene that u desire
use complentart dna with the help of reverse transcriptase to change the rna into complentary dna
how are eukaryotic plasmids introduced to mammalian host cells
via transfection using calcium phosphate or plasmid packaging in liposomes …
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells
what are the agents involved for eukaryotic plasmids?
puromycin or neomycin
why is polymerase chain reaction important
allows amplification and analysis of very small smaples of dna
what are the different things that are used for pcr
fingerprints , cloning genes, infectious diseases, hereditary info ect
what does and doesn’t the reverse transcriptase polymerase chain reaction do (RT-PCR) do ?
it checks the relative expression of specific gene products like mrna transcription but not the actual
how does reverse transcription pcr work
take the gene and isolate it. then use reverse to encode into the dna … then with help of primers to identify the complentary dna strand of the mrna
what is quantitative polymerase chain (qPCR), what is it performed on, and how is it detected?
finds the absolute number of the copies or the relative number normalized to the control.
can be performed on either dna or cdna
detected through fluorescent that will allow dna to bind or through flurescent probe
dna sequencing
is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine.
helps with studying health and disease
ribose vs deoxyribose 2nd carbon
hydroxyl on second carbon of ribose
in dna sequencing technique, why is the third hydroxyl group missing
to prevent elongation of dna strand to occur
describe the dna sequencing
- get sample dna and denature it
- mix in 4 separate test tubes aand add a different dideoxybase to each tube
- let replication occur and see different fragments through electrophoresis.
the bottom starands are the ones farthest from the wells
3.
genomic sequencing and what two approaches it can use?
help to tests cancer by generating
a genetic map linkage with several hundred markers per chromosome where it has a large library of chromosomes but gets smaller and smaller as more cloning takes place
shotgun that skips the gene mapping and cuts the chromosomes into fragments
to convert lactic acid back to pyruvate, u need what molecule
o2
polymorphisms
stretches of repeteitive highly variable dna
2-100 bp
two types of analysis used for dna fingerprinting
restriction fragment length polymorphism
—- includes using restriction endonucleases to cut the polymorphisms into dna fragments that vary in size which is unique to humans
separated using gel electrophoresis and dna south blott
short tandem repeat
uses pcr to amplify polymorhisns that are found in non coding introns
exome and targeted sequencing benefit
only target genes of interest through sequencing only the exons
flurorescence in situ hybridization (FISH)
uses fluorescent probes to locate the positions of specific dna sequences which helps to see presence or absence of specific dna sequences
microarrays
used to study relative rna amounts btween two samples or to compare one to the standard reference.
steps to microarrays
place fluoresxent dye
place on chip that has the binding site for every gene
what does the chip do in micro array?
has the binding site for every gene that acts as a probe to determine transcript levels in tissue samples
when it comes to the respire sys, what relaxies it
sypathet
in situ hybridization (ISH)
similar to fish but examines examines gene of interest in a tissue that is fixed to keep transcripts in place
Immunohistochemistry (IHC)
needs an antibody that is recognized by a second antibody which is either linked to an enzyme or a fluroscent molecule (staining)
used to identify breast tumors
flow cytometry
single cells stained for certain protein markers using specific antibodies linked to fluorescent tags that can emit light
what does the scattered and emitted light show in flow cytometry?
gives info on cell size and how many cells in the sample express each of the markers that were labeled
describe immunoprecipitations
commonly used to find protein binding partners
includes cell lysates, antibody specific for protein of interest
what happens in immunoprecipitations
antibody has bead that its covalently attached to which can be pulled out of soln or precipitated
use a lysate to wash the undesired proteins away
mass spec vs western blot when it comes to protein detection
use western when u have an idea of the protein u are looking for. if u don’t, use mass spec
subcellular location
find the location of the protein by using an expression vector tgat traces the protein with green flurescence
why is altering gene expression useful
it can help determine function of protein by knocking out/in genes , exciting or inhibiting , or looking at the phenotype
in vitro mutagenesis
clone a gene, mutate it, place back into a cell to see alter the gene fuction that u are studying
what are stem cells and do they use mitosis or meiosis
undifferentiated cells that differentiate to become other cell types ..
replicate by mitosis
what are pluripotent cells and give an ex
able to differentiate into any of the three germ layers
ex: embryonic cells
what are adult stem cells used for and give 3 ex of sources
tissue repair and regeneration
ex: bone marrow, adipose tissue, blood
sugars have l or d
carbohydrates (sugars are deliscious)
difference in probes btween western vs northern and southern
antibodies are used in northern and southern for probes… in western they nucleic acids
gene therapy
genetoic disorder is treated bhy introducing to a cell
uses genetically modified virus… retro and adeno viruses
affinity chromatography
biochem mixture that ius based on interactions:
purify proteins
antigen and antibody
enzyme and substare
receptor and ligand
protein and nucleic acid
turning region
loose unstructured region that is not in the alpha or beta structures
what is wrong about glycine?
its so smalls so it disrupts secondary structure
why is a third ionization energy higher than first and second?
3rd electron is being removed from a complete full subshell
which aa have chiral carbons in side chains?
threonine and isoleucine
bases can cut enzymic activity ?
bases can cleave peptide residues
