Molecular Genetics
Final Review Questions
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What is this tool used for?
Discuss the significance of lambda host restriction to the field of molecular genetics.
Distinguish between de novo and hemi-methylation.
Explain how bacteria protect themselves from the restriction enzymes they constitutively produce.
Describe how a restriction enzyme finds it's target sequence.
How does methylation affect this process?
Calculate the average fragment size produced by 4-mer, 6-mer and 8-mer enzymes.
Calculate how long a sequence needs to be if it is to occur only once in
the lambda genome (50,000 bp)
the E. coli genome (5,000,000 bp)
the H. sapiens genome (3,000,000,000 bp)
What is this tool used for?
What is the 'driving force' behind gel electrophoresis?
Explain DNA's constant charge to mass ratio.
If every base pair experiences the same driving force, why to the molecules separate according to size?
Outline the properties of ethidium bromide that make it a very sensitive gel stain for DNA.
What is this tool used for?
Plasmid Vectors
What is an 'episome'? How is it different from a chromosome?
What are the key features present in the plasmid pBR322?
What are the key differences between the plasmids pBR322 and pBluescript?
Describe the positive and negative selections systems you would use to identify recombinants
if you were cloning into the BamHI site of pBR322
if you were cloning into the BamHI iste of pBluescript
What is the significance of intragenic complementation to pBluescript screening?
Lambda Vectors
What are lysogens and transducing phage?
What is their significance to the development of lambda cloning vectors?
Distinguish between lambda insertion and substitution vectors.
Distinguish between recombinant and non-recombinant viruses.
Outline how the lambda cI gene is used to identify and select for recombinant phage
in a mixture of recombinant and nonrecombinant viruses.
How does the HFL mutant affect the lambda life-cycle?
Outline the selection system used to selectively propagate recombinant viruses when using substitution phage.
What is a P2 lysogen?
Differentiate between theta-mode and rolling circle mode lambda replication.
Discuss the use of the 4-mer SauIIIA in the preparation of random 15-20 kb fragments for the construction of 'complete' genomic libraries.
How many recombinants carrying 15 kb random inserts would be required
for 99% coverage of the human genome (N = 3,000,000,000 bp)?
Discuss the use of oligo-dT and random primers in the synthesis of cDNA populations.
Explain why gels used to size fractionate RNA molecules typically contain either 1M formaldehyde or 6M urea.
Define Tm. What is it a measure of?
Tm = 81.5 + 0.41 %GC + 16.6 log [Na+]
Why is there a %GC term?
Discuss the [Na+] term.
An older version of the formula included a term for % formamide included in the sample.
What effect would you expect formamide to have on the Tm of the DNA.
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