exam 3 Flashcards
What can we assume about the ros and anL genes?
That they are on different chromosomes
What is the phenotypic outcome of the F2 group resulting from a F1 (ROS/ros; ANL/anL) self cross?
9/16- 56.25% tall and purple
3/16- 18.75% tall and green
3/16- 18.75% short and purple
1/16- 6.25% short and green
What is Sanger sequencing?
Using DNA polymerase to obtain a relatively short copy
What type of termination is used in Sanger sequencing?
Di-deoxychain termination
How are ddNTPs modified?
With fluorescent tags that emit and label/identity at the end of each fragment
What is added to a Sanger sequencing reaction?
DNA template, primer, DNA polymerase, dNTPs, and ddNTPs
How many primers are added to Sanger sequencing?
One
Why cant you add dNTPs to ddNTPs?
ddNTPs do not have an oxygen on 2’ or 3’ carbon. 3’OH is required to add bases
What is the result of Sanger sequencing?
A large amount of fragments of differencing sizes that only vary due to one base pair
What strand is labeled in Sanger sequencing? Synthesized or template?
Synthesized has the labeled terminal nucleotides and it is complementary to the template
Can you see the nucleotide bases on agarose gel?
No
How is the sequencing chromatogram obtained?
Laser is sent through strands and through a detector
What are the five requirements that tell you that you have a good chromatogram sequence?
- Individual, well defined peaks
- No overlapping peaks unless heterozygosity is expected
- No N bases in the middle of the sequence
- High signal to noise ratio
- 700-1000bp or the length you expect
What does the primer provide for DNA polymerase in the sequencing reaction?
3’ OH group
What is mendels second law?
Principle of independent assortment- gene pairs on different chromosomes pairs assort independently at meiosis
How do you obtain a phenotypic 9:3:3:1 ratio?
F1 self cross
What is the genotype and phenotype of the F1 plants we made in class? What gametes (4) do they produce?
Genotype- ROS/ros; ANL/anL
Phenotype- tall x purple
ROS;ANL
ros;anL
ros;ANL
ROS;anL
What phenotypic ratios do we expect in the F2 progeny?
Tall green R_;aa 3/16 18.75%
Short purple rr;A_ 3/16 18.75%
Tall purple R_;A_ 9/16 56.25%
Short green rr;aa 1/16 6.25%
What is the null and alternative hypothesis for Mendelian segregation chi squared?
Ho: The numbers of plants we observe in the phenotypic classes are consistent with Mendelian segregation
Ha: The number of plants we observe in the phenotypic classes are not consistent with Mendelian segregation, another form of inheritance
What is the purpose of the chi square test?
To compare the observed frequencies of phenotypes vs the expected. Allows us to accept or reject the null hypothesis based on likelihood ratio
Do our expected values need to be mathematical or exact?
Can be mathematical with decimal points
How do you calculate the degree of freedom?
Number of phenotypic possibilities - 1
What P value is used in chi square analysis? What does it demonstrate?
0.05. Demonstrates that there is 95% confidence that the data is not random but real
If the chi square value is less than the critical value? Above?
If less than, accept null hypothesis. If above then reject null hypothesis
How do you create a consensus sequence?
Overlay forward and reverse primer sequence. There will be a region of overlap
Do you need to reverse complement the reverse sequence for consensus sequence composition?
Yes
What is the expected length of the consensus sequence on average?
800-900bp
What is the length of the rbcL and atpA regions we amplified?
1300-1400bp
What does it mean to genotype something?
To determine and compare genetic composition. Associate genotype to phenotype
What are three reasons why we need to use at least 1000 bp of sequence to construct phylogenetic trees?
- need long enough to calculate the percent differences along sequence
- published literature uses 1000 bp
- shorter sequence increases chances of selecting bias in base change
What are the characters we use in tree building?
Can be any binary characteristic but we are using DNA sequences
What is an outgroup?
A taxon outside the group of interest that is known from other evidence to be closely related to that group
What is parsimony?
Grouping taxa in ways that minimize the number of evolutionary changes that have occurred in the characters. Minimal changes are most likely
How do we consider each base in parsimony relationships?
Each base is considered individually
What is maximum likelihood?
mathematical methods for building trees when something is known about the process of evolution in the underlying data set
Does parsimony consider knowledge of evolution?
No
What do we assume about base pairing in maximum likelihood?
A to G and C to T changes are more likely than A to T or C
What is bootstrapping?
Multiple, theoretical resampling of the sites in the alignment used to build new trees
What does scoring mean in bootstrapping?
A score closer to 100 means it is more significant because if you were to repeat the comparison 100 times thats how many times that alignment would occur
What program do we use to build trees?
MEGA
What are the 3 primer pair combinations that are considered standard when setting up a genotyping reaction to test for a mutation in an organism that is represented by a putative insertion?
gene specific, insert/gene hybrid, and insert specific
If a gel has a band for gene specific, what can we assume about it’s genotype?
AA. There is no a strand with an insertion that would produce a band
If a gel has bands for gene specific, insert specific, and insert/gene specific what can we assume about its genotype?
Aa, insertion in a allows for bands with insert specific primers
If a gel has bands for insert specific and gene/insert specific what can we assume about it’s genotype?
aa. Gene specific alone will not work since it does not have A allele
When reading an audio radiographs from Sanger sequencing what can we say about its directionality?
Small bands at bottom are 5’ of synthesized stand and large bands at top are 3’
Why didn’t we use gene/insert specific primers for the anL locus?
It’s insertion is an inverted repeat sequence so after primer binding and cooling it binds to itself and polymerase cannot add new bases
When there is an mutated insertion in a gene, when you use PCR do the primers amplify it only, the intended product only or both?
Both, it is within the sequence you want to amplify so the whole thing will amplify
Would you get a band if you are amplifying a product with an insertion alone?
No since the extension time is set for the wild type band and not the mutated one
What percent gel was used for genotyping analysis gel? Why?
2% gel is used, it increases the ability to visualize the bands since they are small
What is the expected gene specific band length? What genotypes does it associated with?
215bp without inset, can amplify wild type A so see it in AA and Aa
What is the expected insert/gene hybrid band length? What genotypes is it associated with?
265bp, insert is 50bp long. Associated with Aa and aa since it has the a allele with the insert that can be amplified
