Question

You PCR amplify a 500 bp (base pairs) piece of DNA that has diagnostic value in determining whether a patient has a mutation within a specific DNA region. You know that this DNA segment of the “normal” gene does not include an EcoRI restriction site; but the mutated DNA segment of the same gene contains an EcoRI restriction site due to a point mutation at the 100th bp from the 5’ DNA end. After PCR amplification, you subject your DNA to EcoRI digestion, and separate both, the undigested and digested DNA samples by gel electrophoresis.

a.) Draw a schematic of DNA segments for both normal and mutated DNA

.b.) Draw the results of the gel electrophoretic separation after EcoRI digestion for normal (lane 1) and mutated DNA segment (lane 2) assuming the patient carries the mutation only on one parental allele

Marker 1 2 1000 bp 500 bp 200 bp 100 bp

c.) Draw the results of the gel electrophoretic separation after EcoRI digestion for normal (lane 1) and mutated DNA segment (lane 2), assuming the patient carries the mutation on both parental alleles (refer to above image)

Answer 1

a) Schematic representation of DNA fragments for both mutated and normal DNA are shown in the image below:

Normal DNA .500 bp - اد 400 bp Mutated DNA & aar SC GAATTCE еттері C TTA AIGT il 99 — -99- be 400 bp

b)

Marker Marker Jooo be 10обУР Soo LP | Thoob P 500 bp oo be 2ooъг 100 bp Тоо ьp

There are two cases for the electrophoresis separation where patient caries mutation only on one allele.

Fig (i) indicates the Electrophoresis separation for the DNA sequence taken from the mutated allele. Since, restriction site for EcoR I is present due to point mutation and Hence two bands are visible for two fragments. (one at 100bp and other at 400bp length of DNA).  

Fig (ii) indicates the electrophoresis separation for the DNA sequence taken from the normal allele. (Since, no restriction site available for EcoR I).

c)

Marker Unca 100obet 500 bp Phoob P 200 be lovbpta

For DNA sequence taken from patient carrying mutation on both parental alleles, The electrophoresis bands will always be obtained as shown in the above image.