Genetics Lab Exam Flashcards
Lab 1: probability
Likelihood of a single event occurring
Product rule
Probability of 2 independent events being both occurring simultaneously is the product of their individual probabilities. “AND“
Sum rule
probability of either one or the other of two mutually exclusive events occurring is the sum of their separate probabilities. “OR”
Chi square
= total (obs - exp)^2 / exp
Likelihood that 2 sets of results are similar enough to consider them equivalent
Degrees of freedom
takes into consideration the number of independently varying Parameters.
Df= # classes - 1
Use DF for x^2
Chi square significant test results
If statistical test indicates that the difference between expected and observed results is significant then your null hypothesis (hypothesis of ‘no difference’) must be rejected.
X^2
greater the difference between the expected and observed values, the greater the X2 value and the lower the probability that this difference is due to random chance. It is generally accepted that a X2 value with a probability less than 0.05 (5%) indicates a low (<5%) probability that the observed values agree with the expected values by random chance. Therefore observed results are considered to be significantly different from expected results.
X^2 (chi square test) uses
Used to statistically determine whether the null hypothesis should be accepted or rejected
Pipettors
Used to transfer specific volumes of solutions quickly and accurately
Gel electrophoresis
Used to study DNA and it involves separating fragments of DNA for genetic analysis.
Is DNA positive or negative charge
Negative
What is the distance travelled through the electrophoresis gel indicate
- the size of the fragment (smaller fragments will travel further through the gel)
-charge
What is the gel in the electrophoresis made up of
Aragose, distilled water, ethidium bromide (EtBr)
What are restrictions enzymes
Proteins that cut DNA at specific sites. Each cut is made to the phosphodiester bond between specific nucleotides within the sequence. # of cuts depends on # of recognition sequences present in the original DNA molecule. Sequence is usually palindromic (sequence on both strands of the DNA is identical when read in the 5’ to 3’ direction.
Restrictions enzyme digest
Enzymatic manipulation of DNA. Cutting DNA at specific sites. Can be used to shuttle DNA from one organism to another.
One of the first restriction enzymes to be isolated
E.coli (EcoRI)
What types of ends can the cleavage of DNA from restriction enzymes create
Blunt ends or staggered ends
-cut made in middle of sequence usually leaves blunt ends
-cut anywhere else produces complimentary tails on the DNA fragments
Molecular marker or DNA ladder
A protein of a known band size that can be used to compare to your results in the lab to estimate their molecular size
What does assessing molecular weight and # of bands generated by each individual allow for (in a gel electrophoresis)
-identification of the restriction enzyme target sites within the DNA molecule
Restriction map
Map showing the relative locations of restriction sites.
-consists of long horizontal line (DNA molecule) and short vertical lines (cut locations)
How many bands are produced when there are 4 restriction enzyme cut sites
5 bands
In lab 3, what was used to extract DNA from a blood sample (or other biological materials)
Wizard kit
What must PCR reactions contain
Template DNA, free nucleotides, a forward and reverse primer set, and a DNA polymerase to build the new DNA molecules, buffer and magnesium chloride (provides optimal environment for process to occur)
If the DNA evidence matches the DNA from an individual, this evidence is considered to be what type of evidence
Inclusive evidence
-they MAY be guilty but need further evidence
(Forensics lab) If the DNA in the sample differs from an individuals DNA, what can we conclude (What type of evidence is this)
This individual can be ruled out as a suspect with certainty.
Exclusive evidence
Single nucleotide polymorphism (SNP)
Changes in certain DNA regions that only a very small portion of the population (5%) have to narrow down possible suspects
What do we use to amplify extracted DNA
Polymerase Chain reaction (PCR)
What does nucleic lysis do
Nucleic lysis solution breaks down cells and nuclei
What does proteinase K do
proteinase K enzyme breaks the bonds between the amino acids in the proteins.
At what temperature does proteinase K function at best when getting ready to run a PCR
55%
T/F: PCR is extremely sensitive to any amount of DNA
True
A single cell can contaminate the PCR
What type of fish was used in lab 4 (Hardy Weinberg lab)
Chinook salmon
Probability equation
times event A occurs / total # of events
If you see AND with probability question…
Multiply
If you see OR with probability question…
Add
Hypothesis of no effect
Null hypothesis
If null hypothesis if found to be false, then what kind of hypothesis is found to be true?
Alternative hypothesis
Why do we use loading dye in gel electrophoresis
-Adds density to samples (without it, samples are more likely to float out of well, pulls them down into well)
-adds colour so that we can see the solution in the well
Where is the ethidium bromide in the gel electrophoresis
It is added to the gel
-it interacts with the DNA
-what allowed us to visualize the band in the gel images made
What end do we load a solution in a gel electrophoresis
Negative end
What direction does the DNA move in the gel
Towards the Positive end (bc DNA is negative)
How do you differentiate between large vs small molecules in the gel electrophoresis
Largest stay at top (near well)
Smallest are closest to the bottom
If using a P20 pipette, what is max amount of solution it can hold
20 micro litres
What is the DNA polymerase that is often used in PCR called
Taq
(Enzyme extracted from bacteria in deep ocean hydrothermal vents)
Is this sequence palindromic?
5’ GGATCC 3’
3’ CCTAGG 5’
Yes
Name for bacteria that are extremely heat tolerant
Thermophilic (ex. Taq)
Polymorphic nucleotides
Variable among individuals
What are primers
Short sections of single-stranded DNA
Microsatellite DNA
DNA composed of short tandem repeat (STR) polymorphism or highly repetitive elements
Microsatellite genetic analysis (genotypic) is commonly used for 3 purposes in population and conservation genetics…
1)identifying separate populations of organisms
2) identify parentage,
3) to measure genetic diversity
What are attributes that make a marker useful for population analysis…
Clear bands on gel
Correct number of bands
Some variation among individuals
Repeatability
In lab 4, what does 1 band in the gel represent
Homozygote
In lab 4, what does 2 bands in the gel represent
Heterozygote
Types of genetic analysis
HWE
Inbreeding coefficient (F)
Mean fixation index (mean Fst)
Inbreeding coefficent
(F)
Level of inbreeding in each of the populations
Mean fixation index
(Mean Fst)
which is the average genetic divergence between the two populations based on FST values.
How is HWE tested in lab 4
HWE is tested by comparing the expected number of each genotype with the observed number of each genotype using a Chi-square test.
Reflexes genetic stability of the population
What Fst value would you expect two totally different species to have
Greater than 0.40
What type of fish were used in lab 4 (microsatellite lab)
Chinook salmon
Wild population
Farmed population
Inbreeding coefficient (F) calculation
F = (HE - HI) / HE
where HE is the expected heterozygosity based on HWE (=2pq) and HI is individual heterozygosity (= total number of heterozygotes / total number of individuals).
Fixation index (Fst) calculation
Fst = (HT - HS) / HT
Where HS = (2(pfqf) + 2(pwqw)) / 2
where p and q allele frequencies (large and small bands respectively) are calculated for the wild (e.g. pw) and farmed (e.g. pf) populations using each marker. Thus HS is the mean across the two populations.
Where HT (total population heterozygosity):
HT= 2p’q’
where p’ is allele frequency for the large band calculated using all data (i.e. the two populations combined) and q’ is the allele frequency for the small band calculated using all data combined.
After each round of PCR, what happens to the amount of DNA we have
You are doubling amount of DNA we have after each round of PCR
3 steps of PCR process
1) denature
2) annealing
3) elongation
Step 1 of PCR (denature)
2 strands of DNA separate from one another, become 2 separate strands.
Step 2 of PCR- annealing
Primers then come bind to DNA at 5’ end
Step 3 of PCR: elongation
-amount of DNA is doubling each round
-Taq builds in the opposite direction onto the single strand
Reasons transgenic animals are less common within commercial ventures
1) transgenic animals are technically more difficult to make and rear
2) there is stronger public resistance to transgenic animals
Who’s lab did we get fish from in lab 5 (transgenic fish)
Dr. Devlin’s lab
What type of trangenes did dr. Devlin use for his salmon
Growth hormone transgene
What is Dr. Devlin’s method of producing transgene salmon
-Devlin used the Sockeye type I growth hormone gene (from Sockeye salmon) coupled with the metallothionein-B promoter and microinjected it into the blastodisc region of a fertilized Coho salmon egg.
-The metallothionein-B promoter is a very active promoter, causing transcription of the growth hormone gene to be virtually unregulated (i.e. in a ‘turned on’ state).
-Consequently, Devlin’s growth hormone- transgene (“GH-transgene”) Coho salmon are approximately 11 times larger than non- transgene fish and have over 40 times the circulating levels of growth hormone
Possible Side effects of GH-transgene (used in Dr. Devlin’s lab)
-overproduction of cartilage (thus the shape of the GH-transgene fish may be subtly different from the control)
-breakdown of developmental stability (may shower higher asymmetry due to developmental pathways being damaged by epistatic effects with the introduced GH-transgene)
What does isopropanol do
Precipitates DNA
What does ethanol do
Removes impurities
At what stage were the coho salmon from dr. Devlin’s lab injected with the transgene
Egg stage
Extraction of DNA used in lab 5 (transgene fish)
Used “Extract-N-Amp SIGMA)
-extraction of DNA (followed by PCR) in very short time
-cannot store DNA from this bc it is unstable
What is the name given to an organism that contains a gene from a different organism
Transgenic organism
Which species does DR. Devlin rear in his fish farm for sampling
Coho salmon
What component in the PCR reaction is used to ensure only the fish with the GH-transgene amplify (Lab 5)
Primers
Inbreeding coefficient value chart
0-0.2 = low level of inbreeding
0.2-0.5 = moderate level of inbreeding
0.5-1.0 = highly inbred population
Genetic divergence value chart
0.02-0.05 = slight genetic differentiation
0.05-0.15 = moderate genetic differentiation
0.25 + = great genetic differentiation
What are mosaic fish (lab 5)
A large or small fish that contains the trangene in some of its body cells
Which lab technique amplifies DNA
PCR
What equipment is used to separate solutions by density
Centrifuge
If a a band on the gel from one of the suspects matches 1 of the bands from out lodestar, what type of evidence is this
Inclusive evidence
Which genetic analysis checks for genetic stability in population
Hardy Weinberg equilibrium
What is function of dNTPs in PRC process
Building blocks of newly forming DNA
Role of primer set in PRC process
Short set of nucleotides complementary to DNA flanking the amplifying segment
Role of MgSO4 and buffer i PRC process
Provides optimal environment for PRC process
if PCR tube contains 40 double stranded DNA prior to running PCR, How many copies are produced after 6 cycles
2560
If a circular piece of DNA has 3 recognition sites, how may fragments will that restriction enzyme cut the DNA
3
How may degrees of freedom in chi square test with 3 groups
2
If What type of cross would you perform if you want to study principle of segregation
Monohybrid
Why are enzymes (such as EcoRI) important to the survival of a host cell
Ca help stop viruses y cleaving invading DNA ad prevents replication
What happens if you leave your restriction digest solution on ice instead of in heat block
Digest will not occur bc heat is necessary to catalyze the reaction
DNA molecule has 9 recognition sites for enzyme to cut. How may fragments will result
10
How do P and P’ differ
P’ = alleles from wild ad farmed population
P = alleles from just one of the 2 populations
