Question

Although multiple molecules are important in modern molecular labs, in Genetics Lab, we will be focusing on DNA. Throughout the semester, our treatment of the DNA can determine if we are successful or not in meeting our experimental goals.  

You are going to consider several treatments that might affect DNA shape/form/function. We use some of these treatments on purpose in order to manipulate our DNA, but sometimes we avoid these treatments to keep from harming our DNA.

You will be considering these treatments across three types of DNA:

1. pUC19 - pUC19 is a closed-circle plasmid (2686 bp) which used in molecular cloning
2. Lambda phage - Linear DNA from a bacteriophage (viruses that infect bacteria - in this case E. coli). Lambda phage is approximately 45,000 bps long.
3. Mammalian DNA - Eukaryotic mammalian DNA is arranged in very large linear chromosomes. Although this DNA is mechanically sheared to some extent during extraction, there are still pieces that may be hundreds of thousands of bps long.

You will consider what the following treatments could do to the DNA listed above:

1. Severe agitation - aggressive mixing with a votex machine
2. NaOH - treatment with a base
3. High heat - heating to 100^oC
4. Nuclease treatment - treatment with endo- or exonucleases

Think about the structure of the DNA. Hydrogen bonds are holding the bases together, while phosphodiester bonds are holding the sugar-phosphate backbone together.

• Which treatments will break the hydrogen bonds (and denature the DNA-reduce it from double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA)?
• What treatments might break the DNA into pieces (fragmentation).
• Do you think that all of the types of DNA will be affected the same, or will certain treatments affect certain types of DNA more?

Consider the above questions and write out hypotheses for each treatments. Make sure to also address whether or not you think the different types of DNA will have different results.

Answer 1

1. the hydrogen bonds of all type of DNA, a plasmid, genomic and lambda DNA can be broken just by heating at 100 degree celsius temperature.

2.in we vortex or agitate the larger DNA like mammalian DNA vigorously, it may be broken down into smaller pieces but smaller DNA like a plasmid or lambda DNA are less affected by vortexing.

3. if we talk about the effect of various treatment on the different DNA -

a. NaOH treatment - affects the larger and linear DNA molecules it denatures the genomic and lambda DNA but the plasmid DNA (pUC19) is not affected due to its supercoiled structure.

b. high heat 100 degree temperature melts or denatures - break the H bond in all DNA whether it is small or large genomic DNA.

c.Nuclease treatment. nuclease may be of two type exonuclease and endo nuclease. exonuclease affect the linear DNA with open ends like lambda DNA and broken genomic DNA. while endo nuclease cut or cleaves the DNA from internal site. plasmid remains less affected due ti its shape.

d. severe agitation will break down the larger DNA into small pieces so genomic DNA will be affected most.