DNA Sequencing and Fragment Analysis

Restriction mapping was one of the earlier methods designed to characterize a fragment of DNA. The fragment was cut into smaller fragments using a restriction endonuclease. This is an enzyme capable of recognizing a specific base sequence. Once the region is identified, the enzyme cleaves (cuts) the DNA. It is an effective method used to mark a specific sequence along a region of DNA.

What Are Restriction Endonucleases?

Restriction endonucleases are a group of enzymes capable of cutting DNA into smaller pieces. Each enzyme recognizes a specific sequence that is generally 4 to 8 bases in length. EcoR1 is a popular enzyme that cuts a DNA fragment wherever GAATTC is found. It should be noted that this sequence is a palindrome. That means the sequence is the same for forward and complimentary directions.

Most restriction endonucleases used today originated from bacteria. It is one mechanism microorganisms use as defense against foreign DNA such as bacteriophage. Foreign DNA cleaved into smaller fragments loses functionality and becomes harmless to infected bacterial cells. Each restriction enzyme is labeled from the bacterial species of origin. EcoR1 is an enzyme isolated from Escherichia coli.

Restriction Digest:

A fragment of DNA in solution is treated with a specified restriction endonuclease in a process called restriction digest. One example is treatment of a 5,000 base pair (5kb) fragment with EcoR1. The enzyme will cleave (cut) the DNA fragment every time GAATTC is found in the sequence. For example, the digest generates 5 smaller fragments with sizes 250 bp, 500 bp, 750 bp, 1,500 bp and 2,000 bp. The sum of the fragments equals 5 kb. But, how does a researcher know the smaller fragments have been generated when the DNA size is not visible in solution? Fragment sizes are visualized using gel electrophoresis.

Agarose Gel Electrophoresis:

DNA fragments of different size can be separated on agarose gel in a process called electrophoresis. The solution, with digested DNA added, is loaded on a buffered agarose gel. DNA fragments will migrate towards the positive charged anode when electric current is applied (figure 1).

Smaller fragments move through the gel more quickly than larger fragments so the fragments become separated. Once separation is complete, the DNA is stained with a dye such as ethidium bromide. Different size fragments appear as bands when exposed to ultraviolet light. The size of each fragment is estimated when electrophoresed with a standard ladder of known DNA fragments.

Partial Digest Aids Genetic Mapping

Researchers use a technique called partial digest to determine the order of fragments resulting from a full enzyme digest. A partial digest generally cleaves a DNA fragment on some, but not all, of the sites where the enzyme cut site would be. Partial digest could be performed by reducing the amount of time of digest or amount of enzyme added to the solution. One example would be the generation of 2,750 bp, 2,500 bp, 1,750 bp and 1,250 bp (figure 2)

Because smaller fragments from a full digest are multiples of the fragments from the partial digest, it is possible to determine the order of fragments along the original DNA fragment (figure 3).

Researchers perform restriction mapping along an unknown region of DNA using a combination of restriction endonucleases. It provides a relatively simple method to mark regions along the DNA that could be used in future studies. Unknown fragments that could be cut by these enzymes could also be inserted into a bacteria cell called a plasmid providing known markers (primers) that can be used to determine the entire sequence.

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