Question

Today’s problem:
You perform a Northern blot to detect a particular mRNA from total RNA, and you notice two bands instead of one that you expected. The second, unexpected band migrates faster, and you deduce that it is a shorter version of the long mRNA, by approximately 50 nucleotides. You know the entire sequence of the gene, so you can design new probes to characterize the newly observed mRNA. You suspect that the short mRNA is missing a sequence either from its 5 ́ or 3 ́ end.
  
1. Describe how you would design radioactive probes to perform new Northern blots that could verify from what end, the 5 ́ or the 3 ́, the new mRNA is shorter than the long mRNA. Draw a schematic of where on the mRNA the probes should hybridize.
2. You find that it is the 5 ́ end that is missing from the short mRNA, and you want to determine the exact length of the short version at nucleotide resolution. Describe how the method Primer Extension can address this question (Our textbook, Chapter 5, page 102).
3. YoualreadyhaveclonedthelongmRNAinaeukaryoticexpressionvector,andyou want to delete the sequence missing from the short version to create a new vector to test how the short mRNA is expressed in the cells. Your colleague tells you that you can use PCR based Site Directed Mutagenesis to do that. (Site Directed Mutagenesis is described in our textbook on pages 97-99 but only for creating point mutations). How you will design the Site Directed Mutagenesis primers to generate a deletion in the cloned gene? Draw a schematic of the original vector and where the two new primers will hybridize.

Answer 1

1. The total RNA pool contains many types of RNAs. The three major types being mRNAs, rRNAs, and tRNAs. The mRNAs have a distinct structure consisting of 5' Methyl Guanosine cap, the main sequence comprising of exons and introns and a 3' Poly adenosine tail. Thus, in order to select a particular mRNA from this huge pool of mRNA, probes are designed specifically for the exon+intron sequence.

However, the Nothern blot with this probe has given another band that is smaller, and we have to find out whether it lacks either a sequence from 5' or 3' end. To find this, we can design probes that are complementary to both the sequence and the 5' cap or the sequence and 3' tail.

This will help to pull out the specific mRNA along with the tail portion. If the second band lacks 3'end, upon probing with the 3' probe, northern blot will give a single band and probing with the 5' probe will give two bands. However, if the second band lacks 5' end, upon probing with the 5' probe, northern blot will give a single band and probing with the 3' probe will give two bands.

2. Primer walking refers to the use of overlapping primers to determine the sequence of the segment. Probe for the 3'end can be used to pull ou the sequence. Once the sequence has been concentrated, the primer for the 3' end can be used. An overlapping primer with this primer can then be designed to find the next few base pairs. Then a new primer overlapping with this primer can be designed to determine the sequence of the next few base pairs. This can be continued until the entire sequence is unraveled.

3. The long mRNA has been already cloned into the vector. Thus it will have the 5'end as well. The goal is now to create a new vector with the short mRNA i.e. the one that lacks the 5'end. This can be done by Site-Directed Mutagenesis.

i. Design a forward primer for the first 20-25 nucleotides of the start site of the short fragment. Design a reverse primer for the last 20-25 nucleotides preceding the 5'end of the long mRNA and the part of the vector backbone.

ii. Carry out PCR, the resultant product will be a linear DNA segment.

iii. Circularize it using the Ligation mixture and ligase.