BCHM 270- All Questions100% Solved with Verified Solutions
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Module
BCHM 307
Institution
BCHM 307
They can also be designed to only bind to one version of an allele - allele specific
oligonucleotides (ASO). This can be used with PCR to report on whether samples
contain certain mutations or polymorphisms.
Southern Blotting - Answer Transfer of macromolecules onto a solid-phase membranous
sup...
BCHM 270- All Questions100%
Solved with Verified Solutions
PCR - Answer Used to amplify a select DNA sequence in order to compare genes,
detect sequences, or for forensic analysis. DNA is heated to 95°C to denature the
strands, then cooled to 55-60°C so complementary primers can anneal, then increased
to 65°C so Taq DNA polymerase can synthesize complementary strands.
Allele-specific PCR can determine single-nucleotide polymorphisms (SNP) by putting
primers for wild/mutant alleles and then determining the length of promoted DNA using
gel electrophoresis
Gel Electrophoresis - Answer Method used to separate molecules by size (done by
separating by charge) - often done with restriction digest fragments and PCR products
in order to visualize them. Samples are placed on agarose/polyacrylamide gels and then
an electric field is applied. The larger the molecule, the less distance it will travel. The
gel is stained with fluorescent ethidium bromide in order to visualize the distance of the
molecules.
DNA Probes - Answer Single stranded DNA labelled with radioisotopes or fluorescent
dye, complementary to target DNA. They are bound to the target DNA (hybridization)
in order to identify target sequences.
,They can also be designed to only bind to one version of an allele - allele specific
oligonucleotides (ASO). This can be used with PCR to report on whether samples
contain certain mutations or polymorphisms.
Southern Blotting - Answer Transfer of macromolecules onto a solid-phase membranous
support such as nylon or nitrocellulose. This is done after gel electrophoresis and before
hybridization in order to visualize results of these tests.
Southern blotting refers to DNA blotting.
CRISPR-Cas9 - Answer CRISPR are sequences found in bacteria that identify viral genes
injected by bacteriophages and attack with with Cas9 endonuclease before it can hijack
the cell - researchers use this technology to modify DNA sequences in cells
DNA Microarray - Answer Normal and mutant genes are probed with different
fluorescent compounds and visualized at once to determine gene sequences
DNA Cloning - Answer DNA of interest is cleaved with restriction endonucleases and
then introduced into another organism using a vector (virus or plasmid) in order to
study proteins of interest.
Northern Blotting - Answer Transfer of macromolecules onto a solid-phase
membranous support such as nylon or nitrocellulose. This is done after gel
electrophoresis and before hybridization in order to visualize results of these tests.
Northern blotting refers to RNA blotting.
Quantitative PCR - Answer mRNA is reversed transcribed to cDNA, then thermocycler
measures how much fluorescence is present. Used to investigate gene expression.
,From here, the process is identical to regular PCR
cDNA Microarray - Answer Normal and mutant genes are probed with different
fluorescent compounds and visualized at once to determine gene sequences
Performed with mRNA reverse transcribed to cDNA.
SDS-PAGE - Answer Proteins are unfolded by an anionic detergent (SDS), dyed, and
then run through electrophoresis to separate by size
Antibodies - Answer Antibodies are conjugated with enzymes or fluorescent tags
in order to detect their antigen with high specificity. Used for protein purification,
identification, quantification, or localization within a cell of interest.
Western Blotting - Answer Transfer of macromolecules onto a solid-phase membranous
support such as nylon or nitrocellulose. This is done after gel electrophoresis and
before hybridization in order to visualize results of these tests.
Western blotting refers to protein blotting. They use antigen-specific enzyme-labelled
antibodies to confirm the presence and size of the protein on the gel.
ELISA - Answer Direct ELISA: wells are coated with an antigen of interested and
then samples are tested for antibodies - used to check for immunity
Sandwich ELISA: wells are coated with the antibody, samples of unknown amount
of antigen are added to the wells, secondary antibody is added connected to a
colour substrate to visualize presence of the antigen (quantitative)
, Acidity of amino acids - Answer - All free amino acids have two ionizable groups (can
participate in acid/base reactions): weak acid (carboxyl group) and weak base
(amino group)
- R-groups of acidic and basic amino acids can also act as weak acids/bases
For nonionizable R groups, both carboxylic group (pK1 = 2.3) and amino group in
amino acid act as buffering groups (pK2 = 9.1) by becoming deprotonated
- When one group is protonated and the other is not, the molecule is in its zwitterionic
form
- Isoelectric point (pI): pH where the net charge on a molecule is equal to 0
For ionizable R groups, there are 3 distinct buffering groups: pK1, pK2, and pKR
Henderson-Hasselbalch equation - Answer the relationship between the pH of
solution and the concentration of a weak acid (HA) and its conjugate base (A-)
pH = pKa + log [A-]/[HA]
Ribozymes - Answer enzymes made with RNA instead of globular proteins
Isozymes - Answer multiple enzymes that can catalyze one reaction, with
different regulations and effects
Activation of enzymes - Answer - Some enzymes are inactive (apoenzymes) until
binding to a cofactor, when they become active (holoenzyme)
- A cofactor is a non-protein molecule that performs chemical reactions that amino
acids cannot
- Can be inorganic (metal ions such as zinc or iron) or organic (coenzymes - ATP, NADH,
NADPH, coenzyme A), and are referred to as prosthetic groups when tightly bound
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