Restriction Enzymes Part I: Biotech

Describe any pattern you might see in the upper sequence of bases.

There is no specific type of pattern associated with the upper sequence of bases.

Compare the bases in the upper DNA strand to those in the lower strand. Describe any relationship you can see.

A always pairs with T; G always pairs with C.

Now look at the upper sequence of bases and compare it to the lower. Do you notice any grouping of bases that when read toward the right on the upper strand and read to the left on the bottom strand are exactly the same?

CTTAAG.

A restriction enzyme cuts between G and A in the palindromic sequence:

GAATTC.

How many base pairs are there to the left of the cut?

4

How many base pairs are there to the right of the cut?

10

Counting the number of base pairs, is the right fragment the same size as the left fragment?

No, it is larger.

How could you describe the fragment size in reference to the number of base pairs in the fragment?

Fragment 1 is a 4-base-pair fragment.

Fragment 2 is a 10-base-pair fragment.

If the GAATTC palindrome is repeated four times on the same piece of linear DNA, and the restriction enzyme that recognizes that base sequence is present, how many DNA fragments will be produced?

5

If the GAATTCpalindrome repeats are randomly spaced along the DNA strand, then what can you say about the size of the fragments that will be produced when the DNA is digested with a restriction enzyme that recognizes that sequence?

Random sized fragments will be produced.

Describe the appearance of the DNA in solution.

The DNA is colorless.

Is the DNA visible?

No. DNA in solution is not visible.

In which tube do you expect no changes to occur—that is, no DNA fragments produced?

The DNA in the uncut lambda DNA tube, which gets no enzyme, should remain intact as a single band.

What is missing in that tube that leads you to that decision?

No restriction enzymes are added to that tube, thus no digestion will occur, and no fragments will be produced.

This tube (L) is the control tube.

Compare tube P to tube L; What do you expect to happen in the P tube compared to the L tube.

The P tube contains the restriction enzyme PstI. There should be an enzymatic digestion that occurs in that tube, which results in the production of restriction fragments. The L tube only contains DNA and no enzyme and so should not produce any restriction fragments.

Why do you expect this difference?

Restriction enzymes digest DNA at specific sites. If lambda DNA contains restriction sites for the enzyme PstI, then the DNA should be cut into fragments. With no added enzyme, no digestion should occur.

If the DNA in tube L becomes fragmented at the conclusion of the reaction, what can you conclude?

Most likely a restriction enzyme was inadvertently added to the L tube. This could have happened by accidently adding an enzyme to the tube, or by not changing pipet tips, which could result in enzyme being carried over between tubes.

Is there any visible change to the DNA after adding restriction enzymes?

No. The DNA still appears colorless.

If a DNA molecule has two restriction sites, A and B, for a specific restriction enzyme, how many fragments would be produced, if it is cut by that enzyme?

3

Which fragment would be the largest?

Fragment 3.

Which fragment would be the smallest?

Fragment 2.

Draw a DNA molecule that has five randomly spaced restriction sites for a specific palindrome. How many fragments would be produced if they were each cut by a restriction enzyme?

6

In this diagram A and B are different palindrome sequences on a DNA strand. Only the restriction enzyme that recognizes site B is present.Explain why only two fragments would be produced.

The enzyme would cut only at site B, producing two DNA fragments.