miniPCR bio
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Chopped! Using CRISPR/Cas9 to cut DNA - Student's Guide
Version: 1.0 - Release: May 2022 - © 2022 by miniPCR bio™
Student's Guide
P./31
Using mathematical thinking
The CRISPR/Cas9 system is highly specific because the gRNA targets a 20-base region. This
makes it unlikely that the gRNA will recognize more than one target in a given genome. Restriction
enzymes are another class of nucleases that scientists have long used to modify DNA. Restriction
enzymes typically target a specific DNA sequence that is only 4 to 8 bases long, making them
far less specific than the gRNA targeting possible with CRISPR/Cas9. Here we will compare
mathematically the difference in specificity between the CRISPR/Cas9 system and restriction
enzymes.
Probability of a specific DNA sequence occurring
The product rule states that you can calculate the probability of a series of independent events by
multiplying the probability of each event. The product rule can also be applied to calculate the
probability of any DNA sequence occurring. There are four possible DNA bases (A, T, C, and G). If we
assume all four bases are equally likely to occur, the probability that any specific DNA sequence will
occur is (1/4)
n
, where n is the length of the specific sequence.
For example, say you wanted to know the probability of the two-base DNA sequence AT occurring.
The likelihood of having an A in the first position is 1/4 and a T in the second position is also 1/4, so
the probability of the two-base sequence AT occurring in any two-base-pair stretch would be:
(1/4) x (1/4) = (1/4)
2
= 1/16 or a 1 in 16 chance
1. Let's assume we have a restriction enzyme that targets the six-base sequence CGATCG.
What are the chances of this specific six-base DNA sequence matching any random six-base-
pair stretch of DNA? You should use a calculator, but show your work.
What are restriction enzymes?
Restriction enzymes are a class of nucleases that can recognize a specific DNA sequence and
cut both strands of the DNA at that site. Each unique restriction enzyme recognizes a specific
DNA sequence, usually 4 to 8 bases long. Similar to Cas nucleases, restriction enzymes are found
naturally in bacteria and archaea as a defense mechanism against invading viruses, where the
enzymes recognize and cut up the viral DNA. There are thousands of different known restriction
enzymes, many of which are commonly used in the lab to cut DNA.
