BCH5413 Test Questions Solved 100% Correct | Latest Update
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BCH5413
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BCH5413
In the development of knockout mice, it is critical to genotype the mice to
determine if the gene of interest is actually knocked out. Below is a figure for the
primer locations for triplex PCR. Which of the following PCR results would
represent true knockout mice? - Answer One band of relativel...
BCH5413 A Graded Exam with Questions and
100% Correct Answers
Denaturation/Melting of DNA - Answer - separation of DNA strands
- can happen due to temp, salt concentration, G---C content, and length of DNA
(Hyperchromicity effect)
- as temp increases, absorption of light increases due to bases becoming less shielded
when they melt apart
The (greater/lower) the G+C content and the (higher/lower) the salt, the more stable
the DNA duplex - Answer greater G+C and higher salt
What are higher stringency conditions - Answer high heat and low salt
What else can melt DNA? - Answer pH
DNA renaturing and hybridization - Answer - add complementary RNA and re-anneal by
gradually lowering temp.
Where are chemical groups present in the DNA - Answer major and minor groove
How do sequence specific DNA binding proteins bind in a sequence specific manner -
Answer the amino acids of sequence specific DNA binding proteins recognize the
pattern of chemical groups in the major/minor groove
- may interact through different bonding to permit sequence specific recognition
Zinc finger binding proteins - Answer - custom designed DNA binding proteins
- two beta sheets attached to an alpha helix coordinate by a zinc molecule
,- triple zinc finger can bind in the major groove of DNA
Zinc finger custom design - Answer swapping the alpha helix domain and beta-hairpin
segments allows for different sequence specificity
*alpha helix domain determines sequence specificity
Zinc finger properties - Answer Crystallographic studies
- a single zinc finger protein can recognize 3bp of DNA
Zinc finger and DNA binding specifics - Answer - AA of zinc finger at position 6 recognizes
first nucleotide of DNA
- AA of zinc finger at position 3 recognizes second nucleotide of DNA
- AA of zinc finger at position -1 recognizes third nucleotide of DNA
Zinc finger table - Answer helps determine what amino acid sequence of the zinc finger
will match up to what specific triplet sequence of DNA
What is the use of custom designed DNA-binding proteins - Answer potential for
designing a DNA-binding protein to bind to a single site (DNA sequence) in the genome
RNA structure - Answer - single stranded
- 2' OH
- phosphate sugar backbone (uracil not T)
- 5' to 3' orientation
RNA secondary and tertiary structure - Answer significant secondary and tertiary
structure from intramolecular base pairing interactions from different regions of the
RNA
,- watson and crick
- G to U
- others
Eg. stem loop, hairpin, pseudoknot structures
Secondary structure of tRNA - Answer cloverleaf structure
Tertiary structure of tRNA - Answer base pairing and other interactions primarily
between D loop and T-psi-C loop convert "clover leaf" into the "L" conformation
- clover leaf folds back on itself
Differences between types of blots - Answer Southern: DNA
Nothern: RNA
Western: Protein
gel elecrophoresis
- matrix
- gel
- buffers - Answer separation of charged molecules in an electric field
- matrix can be made from agarose or polyacrylamide
- apparatus can be horizontal or vertical
- buffers can be low or high salt, neutral or denaturing
Principal of gel electrophoresis - Answer separation based on size
- DNA restriction fragments are placed in well of gel
, - DNA is negatively charged and will move to the positive side
- the smaller travel more quickly
*rate inversely proportional to their length
What is used to view DNA in gel - Answer ethydium bromide
What is used for digestion of dsRNA.. - Answer use restriction endonuclease(s)
Eg. EcoR1
What is a gel ladder - Answer has bands of a known size
southern blot analysis does what - Answer identify DNA fragments based on their actual
sequence
Steps of southern blot - Answer - starts with electrophoresis of DNA sample
- denature DNA and blot using transfer apparatus (eg. capillary action) onto membrane
- southern blot with invisible DNA bands now on filter
- block membrane with nonspecific DNA or protein
- incubate with labeled probe
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