You run a biochemical analysis lab and you have isolated a 75-nucleotide piece o

Question

You run a biochemical analysis lab and you have isolated a 75-nucleotide piece of mRNA from an E. coli sample you provided. You have determined that 76 percent of this RNA is part of a coding region, while the remaining percentage is the very 5' end of a gene product and hence is non-coding. An HPLC analysis of an RNase-treated aliquot of the mRNA reveals that it contains about equal fractions G, C, A, and U. What is your best guess for the molecular mass of this mRNA? Provide a reasonable extinction coefficient for this RNA for UV absorption at 260 nm and use it to calculate the micromolar concentration of your original sample if a 1/1000 dilution gives an OD_260 of 0.0126 in an Eppendorf Biophotomer with a 1 cm cuvette. Without any further analysis, how much information content (in bits) exists in the nucleotide sequence of the coding region? Similarly, how much information content (in bits) exists in the amino acid sequence generated from the coding region? Describe in as much detail as you can the methods you would use to determine the exact nucleotide sequence of this mRNA based on procedures described in class.

Explanation / Answer

The molecular mass of this mRNA sequence will be approximately 2.6 x104 daltons(where 1 dalton equals the mass of a single hydrogen atom or 1.67 x 10-24 grams).

Associating the OD260 unit with the amount of oligonucleotides present is done mathematically using a physical constant known as the extinction coefficient. It is a component of Beer's Law:

A = Cl

Where A is absorbance, C is concentration, l is the path length, and is the extinction coefficient, a constant for the material being analyzed.

Given A=260, C=0.0126 for 1/1000 dalton=12.6(when converted to 1 dalton), I =1

=260/12.6*1=20.63 OD units/ µmole

Converting to µmoles:

µmoles of oligo = OD (A260) ÷
= 12.6 ÷ 20.63 OD units/µmole
= 0.610 µmoles

The information content in bits for nucleotides in the coding region would be 22.8 bytes (given by the number of nucleotides that code taken as 57 and the bits in one triplet taken as 0.14) and for the amino acid sequence the information content will be 0.14bits, for each triplet.

The last question is related to discussion in class about different procedures so you will have to outline those to refine your answer. One of the methods is to use a NCBI Blast service to perform a similarity search. There are other sites as well that provide services of a similar kind.

Also please refer to links below for reference:

http://eu.idtdna.com/pages/docs/educational-resources/molecular-facts-and-figures.pdf?sfvrsn=4

https://www.trilinkbiotech.com/tech/extinction_intro.asp

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