Biotechnology - Restriction Enzyme Analysis of DNA: Biology Lab

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  • 0:02 DNA & Restriction Enzymes
  • 2:37 DNA Mapping
  • 4:18 Analyzing the Results
  • 5:53 Lesson Summary
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Lesson Transcript
Instructor: Jennifer Szymanski

Jen has taught biology and related fields to students from Kindergarten to University. She has a Master's Degree in Physiology.

As any amateur sleuth knows, DNA is an important part of crime scene analysis. But why is that, and how is it used in the lab? This lesson will explore restriction enzymes analysis of DNA and how it's used to 'fingerprint' suspects in a crime.

DNA and Restriction Enzymes

If you've watched any crime show - ever - you know that DNA is used to place and/or eliminate suspects from the scene of a crime. Have you ever wondered how this thin, twisted molecule can be used to reveal so much about us?

Remember that the DNA 'ladder' is made of three big components: a phosphate backbone, a sugar (deoxyribose), and rungs made of nitrogenous base pairs. Each of the bases pair specifically with one other. A (adenine) pairs with T (thiamine), and G (guanine) pairs with C (cytosine). The ways in which these four little letters can be arranged are almost endless, and the sequence in which they are arranged sets us apart from other people.

Thanks to special proteins called restriction enzymes, we can definitively see how. Restriction enzymes are proteins that digest (cut) DNA at specific base sequences. Although exactly what sequence varies between restriction enzymes, the so-called recognition sites, sequences 'picked out' by the enzyme have something in common - they're palindromes, meaning they read the same forward and backward, just like some words you might know. Since we're looking at two-stranded DNA, the palindrome is a little different; it reads forward on one strand and backward on the complementary (paired) strand.

For example, a restriction enzyme called EcoRI recognizes the sequence GAATTC. Notice its complement: CTTAAG. EcoRI scans the length of the DNA molecule, and every time it finds this sequence, it makes a cut between the G and the A on both strands of the DNA. In most cases, the actions of restriction enzymes result in sticky ends - unpaired bases that hang over. Scientists can 'glue' other, complementary pieces of DNA to these ends with enzymes called 'ligases' to create whole new, tailored DNA sequences. This technique is often used to make medicines used in all kinds of therapy. Depending on how many times this sequence appears in a person's DNA, this results in two or many more fragments of different sizes.

DNA Mapping

Forensic scientists are able to take the DNA fragments that result from digestion by restriction enzymes, now called RFLPs (restriction fragment length polymorphisms), and create a DNA fingerprint. To do this, they use a technology called gel electrophoresis, which literally translates to 'carrying by electricity.'

Gel electrophoresis uses the power of electricity to sort the RFLPs by size. In this technique, the pieces of DNA are pulled through a gel of agarose, which is a sugar present in seaweed. The agarose creates a matrix through which smaller pieces are able to move more quickly and larger pieces move more slowly.

It makes sense: say you are standing on the edge of a very thick jungle, full of tall trees and tangled weeds and other undergrowth. If you place an elephant, a goat, and a mouse at the edge of this jungle, and get them to run into the forest as fast as they can for ten minutes, which animal will get the furthest? The mouse, right? Its small size will enable it to slip through obstacles, while the poor elephant struggles to trample through the brush. And most likely the goat will come out somewhere in between.

This 'race' is visible on the gel as bands. As the DNA molecules' negative backbone is attracted to the cathode, or positive end present at one end of the gel, smaller pieces of DNA get further away from the starting point and larger ones remain behind.

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