Lab Practical Flashcards
the probability of two or more independent events occurring simultaneously = ___
the product of their individual probabilities
if two coins are tossed together, the chance that each will be heads is ___. the chance that both will be heads is ___. the chance for the first coin to fall heads and the second tails is ___. the total probability of obtaining a head on one and tails on the other is ___.
1/2
1/4
1/4
1/4+1/4=1/2
when Aa produces gametes, the probability is that ___ of the gametes will contain the A allele and ___ will contain a. when Aa x Aa, the probability is ___ that an A egg and A sperm come together to produce AA. probability is ___ for Aa and ___ for aa.
1/2
1/2
1/4
1/2
1/4
if four babies are born in a given hospital on the same day, what is the probability that all four will be boys?
1/2 x 1/2 x 1/2 x 1/2 = 1/16
what is the probability that three will be boys and one will be a girl?
1/16 + 1/16 + 1/16 + 1/16 = 1/4
what is the probability that two will be boys and two will be girls
6(1/16) = 3/8
what combination of boys and girls among the four babies is most likely to occur?
two boys and two girls
what is the probability that a fifth child will be a boy? girl?
1/32
1/32
the probability of either one or the other mutually exclusive events occurring = ___
the sum of their individual probabilities
the study of chromosome morphology, structure, pathology, function, and behavior
cytogenetics
an individuals collection of chromosomes
karyotype
karyotypes are used to look for ___
abnormal chromosome structure or number
technique for detecting and locating a specific DNA sequence on a chromosome
fluorescent in situ hybridization (FISH)
FISH relies on exposing chromosomes to a small DNA sequence called a ___ that has a ___ molecule attached to it
probe
fluorescent
what is the image of a chromosome set called
karyotype
T/F: FISH is a form of hybridization
true
T/F: karyotyping involves growing cells prior to their analysis
true
effectively inactivating ___ and preventing them from ___ is a key early step in the DNA purification process
endogenous nucleases (DNase enzymes)
digesting the genomic DNA
four things DNA purification methods must accomplish
- effectively disrupt cells or tissues
- denature proteins and nucleoprotein complexes
- inactivate endogenous nucleases
- purify nucleic acid target away from other nucleic acids and proteins
most purification methods disrupt cells using lysis buffer containing ___, ___, and ___
detergent, denaturants, and additional enzymes
disrupt the lipid bilayer of the cell membrane in DNA purification
detergents
release chromosomal DNA and denature proteins in DNA purification
denaturants
four overall steps of DNA purification
- collect cells
- break open (lyse) cells
- remove proteins
- condense the DNA
to break open the cells, ____ is used to dissolve the membranes in a solution called the ___
detergents
lysis buffer
to remove the proteins from DNA, ___ is added which breaks down proteins
protease
protease is an ___ that works best at ___ degrees. the protease denatures the proteins associated with ___ and helps digest any remaining ___
enzyme
50
DNA
cell or nuclear membrane proteins
___ and ___ are then used to bring the DNA out of the solution, or ___.
salt and cold alcohol
precipitate
match the outcomes with the laboratory steps
A- gently chew the insides of your mouth and then rinse vigorously with water
B- add protease, incubate at 50 degrees
C- mix in detergent solution
D- layer cold alcohol over cell extract
E- add salt
___ harvest the cells
___ dissolve cell membranes
___ precipitate the DNA
___ break down proteins
___ make DNA less soluble in water
A
C
D
B
E
which of the following differs between your DNA and that of bacteria living on your skin:
the specific information contained within the DNA
the location of DNA within the cell
the basic principles of how DNA is replicated
the role DNA plays in making proteins
A and B
what is a double stranded molecule of DNA called
chromosome
T/F: DNA is located inside the nucleus of all cells
false
in a nucleotide, what is attached to the 3’ carbon in deoxyribose?
a hydroxyl group (OH)
to which carbon in deoxyribose is the phosphate group attached?
5’
DNA polymerase always adds nucleotides onto the ___ end of the template strand
3’
there are ___ hydrogen bonds between A and T and ___ between G and C
two
three
which base pair (AT or GC) would be harder to separate
GC
in which direction does helicase move along the DNA
away from the origin of replication
what is the role of helicase
it separates the two strands of DNA by breaking the hydrogen bonds between bases
primase synthesizes ___
RNA
does the leading strand or the lagging strand have more RNA
lagging strand
which enzyme synthesizes RNA primers
primase
which enzyme seals nicks in DNA
ligase
which enzyme replaces RNA primers with DNA
DNA polymerase I
which enzyme extends RNA primers with DNA
DNA polymerase III
which enzyme separates DNA strands
helicase
in PCR, what are primers made of
single stranded DNA
in PCR, where should the primers bind
the ends of the region to be amplified
after every cycle of PCR, how does the amount of double stranded DNA change?
it doubles
why is DNA polymerase I not necessary for PCR
there are no RNA primers to remove
which statement about DNA is false?
DNA is present and essential for all living cells
DNA is located in the nucleus of eukaryotic cells
DNA is always copied perfectly during DNA replication
DNA can be circular or linear
DNA is always copied perfectly during DNA replication
what would be a consequence if a cell were unable to replicate its DNA
the cell would not be able to undergo cell division
one strand of DNA has the sequence 5’ AGC 3’. what is the complementary sequence?
5’ GCT 3’
what does it mean to say that DNA polymerase III proceeds 5’ to 3’
DNA polymerase III adds nucleotides to the 3’ end of a growing strand of DNA
what is the difference between the leading and lagging strands in DNA replication
after extension, the leading strand is continuous, and the lagging strand is composed of disconnected fragments
what is the order of enzymes used in DNA replication
helicase, primase, DNA polymerases, ligase
transcription is performed by ___ and translation is performed by ___
RNA polymerase
ribosomes
RNA polymerase binds to the ___ to start transcription
promoter
can an mRNA molecule be translated more than once
yes
the genes in an operon share a single ___
promoter
where can a repressor protein bind on DNA
operator
how does a repressor block transcription
physically blocks RNA polymerase from binding to the promoter
what is the relationship between a gene and an operon
an operon is a group of bacterial genes that share a promoter
what is a major difference between activator proteins and repressor proteins
activator proteins increase transcription when bound to DNA, but repressor proteins decrease transcription when bound to DNA
suppose you destroyed a bacterial cell’s ability to make cAMP. would this affect the cell’s ability to metabolize glucose and lactose, and if so, how?
yes. the cell could metabolize glucose normally, but could only use a small amount of lactose
what is the mechanism by which CAP (an activator protein) increases expression in the lac operon
CAP binds to its activator site, which helps RNA polymerase bind more tightly to a weak promoter
what is a conditional mutation
the phenotype of the mutation is only observable under certain conditions
the trp yeast strain has a conditional mutation that prevents it from growing in the absence of ___
tryptophan
bakers yeast (S. cerevisiae) has a life cycle where the cells exist in either the ___ or ___ state
diploid or haploid
in the haploid state of these cells, they in in one of two forms of ___ which will be attracted to each other where they will ___
mating types
fuse to form a diploid cell
diploid cells can then undergo ___ where ___ progeny will reappear
meiosis
haploid
yeast mating types are attracted to one another by releasing a ___ that the other can recognize through its expression of a specific ___
pheromone
cell surface receptor
once the receptor and pheromone interact, the receptor activates ___ just under the cell membrane which then activate a series of ___ which ultimately activates a set of ___ which activates a specific set of ___
G proteins
kinases
transcription factors
genes
one of the genes activated is called ___, whose gene product causes the cell to change so it can fuse with the other cell
FUS1
to readily monitor the FUS1 gene, it has been fused with the reporter gene ___ which codes for enzyme ___
lacZ
beta galactosidase
beta-galactosidase can be monitored through the use of the substrate ___, which is cleaved to release a ___ product which can be quantified with a spectrophotometer
ONPG
yellow
the more enzyme (beta-galactosidase) produced, the ___ substrate converted to product
more
most molecular analysis of DNA depends on ___
hybridization
where does green fluorescent protein (GFP) come from
bioluminescent jellyfish
one or more small circular pieces of DNA in bacteria
plasmids
the pGLO plasmid encodes the gene for ___ and the gene for ___
GFP
ampicillin resistance
the gene for GFP can be switched on in transformed cells by adding ___ to the cells’ nutrient medium
the sugar arabinose
selection for cells that have been transformed with pGLO DNA is accomplished by growth on ___
antibiotic plates
transformed cells will appear ___ on plates not containing arabinose, and ___ where arabinose is included in the medium
white
fluorescent green
the goal of genetic transformation is to change ___
an organism’s phenotype
scientists use a process called ___ to insert genes coding for new traits into a plasmid
genetic engineering
three main steps of this transformation
- use a transformation solution of CaCl2
- heat shock
- provide cells with nutrients and a short incubation period to begin expressing their newly acquired genes
four genes on the pGLO plasmid and what they code for
- BLA - codes for betalactamase (cleaves ampicillin)
- GFP - green fluorescent protein
- oriC - origin of replication
- araC - regulatory site, needs arabinose to initiation transcription of genes
what do you expect to see on each plate?
1. +pGLO; LB/amp
2. +pGLO; LB/amp/ara
3. -pGLO; LB/amp
4. -pGLO; LB
- colonies
- colonies that glow
- negative control - no growth
- control - bacteria covering the whole plate
what two factors must be present in the bacteria’s environment for you to see the green color?
the GFP gene and UV light
what advantage would there be for an organism to be able to turn on or off particular genes in response to certain conditions
certain genes (like the fluorescence gene) could make organisms more susceptible to predators
transformation efficiency is equal to ___
total number of cells growing on the agar plate / amount of DNA spread on the agar plate (in ug)
what is bioinformatics
the science of using computational methods to decipher the biological meaning from information contained in an organismal system
regions that look like a gene but are nonfunctional
pseudogenes
beta globin has ___ pseudogenes on chromosome ___
2
11
a mutations for which codon leads to sickle cell disease
codon 6, resulting in glutamic acid being replaced with valine
how many mutations have been identified for beta thalassemia
over 500
five genes found within the beta globin region of chromosome 11
epsilon, G-gamma, A-gamma, delta, and beta
how are the five genes similar
they all function to carry oxygen in the blood
how are the five genes different
timing of expression
epsilon is normally expressed in the ___; A-gamma and G-gamma are normally expressed during ___; beta and delta are normally expressed in ___
embryonic sac
fetal development
adulthood
beta and delta produce hemoglobin with a ___ affinity for oxygen than the other three genes
lower
is intron or exon size more free to evolve. why?
intron; mutations in introns cause no harm, therefore more mutations can occur.
process where a molecule absorbs light at one wavelength and then gives off light at another wavelength
fluorescence
GFP is fluorescent because it contains a ___ portion that fluoresces
chromophore
three steps of producing a pure protein
- get the cells to make or “express” the protein
- break open the cells to let the protein out
- purify the protein from the other cell contents
what did we use to make GFP
E. coli that we transformed with the pGLO plasmid
to break open the E. coli cell wall, the enzyme ___ was used and then the cell membrane was broken with ___
lysozyme
repeated cycles of freezing and thawing
two steps of purifying GFP from other cell contents
- discard any material that is not water soluble
- separate GFP from water-soluble material with chromatography
a term that describes a huge and diverse set of methods for separating molecules based on their different properties
chromatography
three simple steps of chromatography
- binding
- wash
- elution
in the binding step of chromatography, a mixture of molecules is exposed to a ___, the molecule of interest binds to solid via ___, the others do not
solid material
non-covalent interaction
in the wash step of chromatography, the solid is rinsed with ___ which allows the molecules of interest to ___ while the unwanted molecules ___
wash buffer
remain bound to the solid
wash away
in the elution step of chromatography, the solid is rinsed with ___ which allows the molecule of interest to ___. the resulting solution ideally contains ___
elution buffer
release from the solid
only the molecule of interest
the GFP we used has significantly more ___ amino acids on its surface that most proteins
hydrophobic
because GFP is so hydrophobic, it will bind to a solid that has a ___ surface more tightly than other proteins would
hydrophobic
in solutions with a high salt concentration, the hydrophobic effect is ___; in solutions with a low salt concentration, the hydrophobic effect is ___
strengthened
weakened
the solid material used for purification (called ___) was packed into a tube called the ___
resin
chromatography column
what salt concentration is used in the binding step
high
what salt concentration is used in the wash step
medium
what salt concentration is used in the elution step
low
what is a protein
a macromolecule that is made up of amino acids held together by peptide bonds
list three examples of proteins found in your body
collagen, enzymes, hemoglobin
explain the relationship between genes and proteins
genes are transcribed into mRNA, which is translated into proteins
describe cloning
a process of copying or duplicating DNA
describe how bacterial cloned cells on your LB/amp plate differ from cells on your LB/amp/ara plate
bacterial cloned cells on the LB/amp plate will only be resistant to ampicillin, but will not glow under the US light because the plate lacks arabinose. the cells on the LB/amp/ara plate will be resistant to ampicillin and glow under UV light
what is the function of the centrifuge in GFP purification
pellets the bacteria to separate it from the growth media
what is the function of lysozyme in GFP purification
digests the bacterial cell wall before freezing
what is the function of freezing in GFP purification
causes the digested bacterial cell wall and membrane to burst
why does the bacterial cell membrane burst when the cells are frozen
cytoplasm expands when its frozen
what was the purpose of rupturing or lysing the bacteria
GFP had to be released in order to purify it
briefly describe hydrophobic interaction chromatography and identify its purpose in this lab
chromatography is a method of separating proteins. hydrophobic chromatography does this by using the hydrophobic properties of the desired protein to accomplish the goal of purifying it. GFP has hydrophobic properties, so this method was used in this lab.
functions to prepare the column for binding to GFP
equilibration buffer
a high salt buffer that allows GFP to bind to the column
binding buffer
a medium salt concentration buffer used to wash away other proteins while still allowing GFP to stay bound to the column
wash buffer
a low salt buffer that allows GFP to come loose from the column and be collected
TE (elution) buffer
process in which DNA is cut with enzymes that recognize specific sequences and then the fragments are separated on a gel to reveal a specific pattern for an individual
DNA profiling / DNA fingerprinting
enzymes produced by bacteria to cut the DNA of viruses that attack them that are used in DNA profiling
restriction enzymes
different restriction enzymes recognize ___ to cut
different specific sequences
Restriction enzymes cut DNA in specific locations resulting in a unique combination of ___ for each organism
fragment sizes
three steps of DNA profiling
- digest DNA samples with restriction enzymes
- use electrophoresis gel chamber to separate the DNA fragments by size
- use a staining process to stain and visualize the DNA fragments in the gel
to cut DNA with restriction enzymes:
isolated DNA samples are added in a ___ to a microcentrifuge tube. ___ is then added and the sample is centrifuged then incubated in a water bath
restriction enzyme buffer
restriction enzyme mixture
loading dye that contains ___ is added to each sample which helps ___ the samples in the gel and also contains ___ which helps each sample to ___ in the well.
bromophenol
glycerol
a solution of DNA fragments of known size used to compare unknown DNA to estimate fragment size
DNA ladder
do large or small molecules go further in the gel matrix in electrophoresis
small
DNA fingerprinting is often based on ___ analysis
RFLP
How many grams of agarose would you add to 200ml of buffer to give you a final concentration of 2%?
4G
what is needed for PCR
template DNA, DNA polymerase (Taq polymerase), primers, dNTPs, buffer
three steps of PCR
- denaturation
- priming (annealing)
- extension
what happens in the denaturation step and at what temperature does this step occur
hydrogen bonds are broken between nitrogenous bases to separate DNA into two single strands
95C
what happens in the annealing step and at what temperature does this step occur
primers attach to their complementary sites on the separated DNA strands
60C
what happens in the extension step and at what temperature does this step occur
Taq polymerase begins synthesizing new strands starting at the primers
72C
p = ___
the frequency of the dominant allele
q = ___
the frequency of the recessive allele
p^2 = ___
the frequency of the homozygous dominant genotype
2pq = ___
the frequency of the heterozygous genotype
q^2 = ___
the frequency of the homozygous recessive genotype
p + q = ___
1
what is the hardy Weinberg equation
p^2 + 2pq + q^2 = 1
chi squared = ___
(observed - expected)^2 divided by expected
degrees of freedom = ___
number of groups minus one
