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molecular genetics > exam 3 > Flashcards

exam 3 Flashcards

1
Q

What can we assume about the ros and anL genes?

A

That they are on different chromosomes

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2
Q

What is the phenotypic outcome of the F2 group resulting from a F1 (ROS/ros; ANL/anL) self cross?

A

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

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3
Q

What is Sanger sequencing?

A

Using DNA polymerase to obtain a relatively short copy

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4
Q

What type of termination is used in Sanger sequencing?

A

Di-deoxychain termination

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5
Q

How are ddNTPs modified?

A

With fluorescent tags that emit and label/identity at the end of each fragment

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6
Q

What is added to a Sanger sequencing reaction?

A

DNA template, primer, DNA polymerase, dNTPs, and ddNTPs

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7
Q

How many primers are added to Sanger sequencing?

A

One

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8
Q

Why cant you add dNTPs to ddNTPs?

A

ddNTPs do not have an oxygen on 2’ or 3’ carbon. 3’OH is required to add bases

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9
Q

What is the result of Sanger sequencing?

A

A large amount of fragments of differencing sizes that only vary due to one base pair

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10
Q

What strand is labeled in Sanger sequencing? Synthesized or template?

A

Synthesized has the labeled terminal nucleotides and it is complementary to the template

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11
Q

Can you see the nucleotide bases on agarose gel?

A

No

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12
Q

How is the sequencing chromatogram obtained?

A

Laser is sent through strands and through a detector

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13
Q

What are the five requirements that tell you that you have a good chromatogram sequence?

A
  1. Individual, well defined peaks
  2. No overlapping peaks unless heterozygosity is expected
  3. No N bases in the middle of the sequence
  4. High signal to noise ratio
  5. 700-1000bp or the length you expect
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14
Q

What does the primer provide for DNA polymerase in the sequencing reaction?

A

3’ OH group

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15
Q

What is mendels second law?

A

Principle of independent assortment- gene pairs on different chromosomes pairs assort independently at meiosis

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16
Q

How do you obtain a phenotypic 9:3:3:1 ratio?

A

F1 self cross

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17
Q

What is the genotype and phenotype of the F1 plants we made in class? What gametes (4) do they produce?

A

Genotype- ROS/ros; ANL/anL
Phenotype- tall x purple
ROS;ANL
ros;anL
ros;ANL
ROS;anL

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18
Q

What phenotypic ratios do we expect in the F2 progeny?

A

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%

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19
Q

What is the null and alternative hypothesis for Mendelian segregation chi squared?

A

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

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20
Q

What is the purpose of the chi square test?

A

To compare the observed frequencies of phenotypes vs the expected. Allows us to accept or reject the null hypothesis based on likelihood ratio

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21
Q

Do our expected values need to be mathematical or exact?

A

Can be mathematical with decimal points

22
Q

How do you calculate the degree of freedom?

A

Number of phenotypic possibilities - 1

23
Q

What P value is used in chi square analysis? What does it demonstrate?

A

0.05. Demonstrates that there is 95% confidence that the data is not random but real

24
Q

If the chi square value is less than the critical value? Above?

A

If less than, accept null hypothesis. If above then reject null hypothesis

25
Q

How do you create a consensus sequence?

A

Overlay forward and reverse primer sequence. There will be a region of overlap

26
Q

Do you need to reverse complement the reverse sequence for consensus sequence composition?

A

Yes

27
Q

What is the expected length of the consensus sequence on average?

A

800-900bp

28
Q

What is the length of the rbcL and atpA regions we amplified?

A

1300-1400bp

29
Q

What does it mean to genotype something?

A

To determine and compare genetic composition. Associate genotype to phenotype

30
Q

What are three reasons why we need to use at least 1000 bp of sequence to construct phylogenetic trees?

A
  1. need long enough to calculate the percent differences along sequence
  2. published literature uses 1000 bp
  3. shorter sequence increases chances of selecting bias in base change
31
Q

What are the characters we use in tree building?

A

Can be any binary characteristic but we are using DNA sequences

32
Q

What is an outgroup?

A

A taxon outside the group of interest that is known from other evidence to be closely related to that group

33
Q

What is parsimony?

A

Grouping taxa in ways that minimize the number of evolutionary changes that have occurred in the characters. Minimal changes are most likely

34
Q

How do we consider each base in parsimony relationships?

A

Each base is considered individually

35
Q

What is maximum likelihood?

A

mathematical methods for building trees when something is known about the process of evolution in the underlying data set

36
Q

Does parsimony consider knowledge of evolution?

A

No

37
Q

What do we assume about base pairing in maximum likelihood?

A

A to G and C to T changes are more likely than A to T or C

38
Q

What is bootstrapping?

A

Multiple, theoretical resampling of the sites in the alignment used to build new trees

39
Q

What does scoring mean in bootstrapping?

A

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

40
Q

What program do we use to build trees?

A

MEGA

41
Q

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?

A

gene specific, insert/gene hybrid, and insert specific

42
Q

If a gel has a band for gene specific, what can we assume about it’s genotype?

A

AA. There is no a strand with an insertion that would produce a band

43
Q

If a gel has bands for gene specific, insert specific, and insert/gene specific what can we assume about its genotype?

A

Aa, insertion in a allows for bands with insert specific primers

44
Q

If a gel has bands for insert specific and gene/insert specific what can we assume about it’s genotype?

A

aa. Gene specific alone will not work since it does not have A allele

45
Q

When reading an audio radiographs from Sanger sequencing what can we say about its directionality?

A

Small bands at bottom are 5’ of synthesized stand and large bands at top are 3’

46
Q

Why didn’t we use gene/insert specific primers for the anL locus?

A

It’s insertion is an inverted repeat sequence so after primer binding and cooling it binds to itself and polymerase cannot add new bases

47
Q

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?

A

Both, it is within the sequence you want to amplify so the whole thing will amplify

48
Q

Would you get a band if you are amplifying a product with an insertion alone?

A

No since the extension time is set for the wild type band and not the mutated one

49
Q

What percent gel was used for genotyping analysis gel? Why?

A

2% gel is used, it increases the ability to visualize the bands since they are small

50
Q

What is the expected gene specific band length? What genotypes does it associated with?

A

215bp without inset, can amplify wild type A so see it in AA and Aa

51
Q

What is the expected insert/gene hybrid band length? What genotypes is it associated with?

A

265bp, insert is 50bp long. Associated with Aa and aa since it has the a allele with the insert that can be amplified

molecular genetics (5 decks)

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