Restriction Enzymes Overview
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Questions and Answers

What do restriction enzymes do?

Recognise specific sequences within a DNA molecule (often 4-6 base pairs long) and cut them in a specific manner.

What is the natural function of a restriction enzyme?

To destroy foreign DNA entering the cell by cleaving the bacteriophage DNA to prevent infection.

What do type II restriction endonucleases do?

Recognise a specific DNA sequence (often 4 to 6 nucleotides in length) and cut precisely or near this site.

What are some examples of restriction endonucleases?

<p>EcoRI from E.coli (GAATTC, sticky), BamHI from Bacillus amyloliquefaciens (GGATCC, sticky), AluI from Arthrobacter luteus (AGCT, blunt), Sau3A from Staphylococcus aureus (GATC, sticky).</p> Signup and view all the answers

What are blunt ends?

<p>When restriction enzymes cut straight across the double-stranded DNA.</p> Signup and view all the answers

What are sticky ends?

<p>When restriction enzymes cut in a staggered fashion across its recognition site.</p> Signup and view all the answers

What is interesting about BamHI, BglIII and Sau3A?

<p>They recognise different sites but produce the same sticky ends.</p> Signup and view all the answers

How can the average frequency of occurrence of a recognition site be calculated?

<p>A tetranucleotide is found once every $4^4$ times, and a hexanucleotide is found once every $4^6$ times.</p> Signup and view all the answers

How are restriction enzymes used in the lab?

<p>The reaction is carried out in a microfuge tube usually at 37°C for 1 hour with sterile components.</p> Signup and view all the answers

What is agarose gel electrophoresis?

<p>A technique used to separate DNA molecules by mixing agarose with a buffer, heating, and cooling it to form a gel matrix.</p> Signup and view all the answers

What is the method for agarose gel electrophoresis?

<p>Agarose and buffer are heated, ethidium bromide added, poured onto a glass plate, and DNA is loaded into wells for separation.</p> Signup and view all the answers

How can DNA fragments be visualized after agarose gel electrophoresis?

<p>DNA fragments need to be stained with ethidium bromide either during or after electrophoresis.</p> Signup and view all the answers

How can the size of DNA fragments be measured after electrophoresis?

<p>Fragments of known molecular weight are run on the gel to determine the size of unknown fragments.</p> Signup and view all the answers

What is restriction mapping?

<p>A method to determine the relative proportions of restriction sites for particular enzymes in a DNA molecule.</p> Signup and view all the answers

Study Notes

Restriction Enzymes Overview

  • Function: Restriction enzymes recognize and cut specific DNA sequences, typically 4-6 base pairs long.
  • Natural Role: They destroy foreign DNA to protect the cell, cleaving bacteriophage DNA; the cell's DNA is protected by methylation.

Type II Restriction Endonucleases

  • Recognize specific DNA sequences (4-6 nucleotides).
  • Cut precisely or near these recognition sites, often at palindromic sequences.

Examples of Restriction Endonucleases

  • EcoRI: From E.coli, recognizes GAATTC, creates sticky ends.
  • BamHI: From Bacillus amyloliquefaciens, recognizes GGATCC, also produces sticky ends.
  • AluI: From Arthrobacter luteus, recognizes AGCT, produces blunt ends.
  • Sau3A: From Staphylococcus aureus, recognizes GATC, generates sticky ends.

End Types

  • Blunt Ends: Created when enzymes cut straight across DNA; can be rejoined with reduced efficiency due to lack of hydrogen bonds.
  • Sticky Ends: Produced by staggered cuts; the ends can base pair thanks to hydrogen bonding, facilitating rejoining.

Enzyme Compatibility

  • Enzymes like BamHI, BglIII, and Sau3A produce the same sticky ends despite recognizing different sites, allowing fragments to be joined.

Frequency of Recognition Sites

  • A tetranucleotide appears once every 4^4, and a hexanucleotide appears once every 4^6; actual occurrence varies due to non-random distribution.

Laboratory Use of Restriction Enzymes

  • Digestion occurs in a microfuge tube with sterile components, typically incubated at 37°C for 1 hour; EDTA, heat, or phenol can stop the reaction.

Agarose Gel Electrophoresis

  • Agarose forms a gel matrix for separating DNA molecules by size when mixed with a buffer, heated, and cooled.

Agarose Gel Electrophoresis Method

  • Combine agarose and buffer and heat; cool to add ethidium bromide.
  • Pour into a gel former with a comb; remove comb after solidifying.
  • Place gel in an electrophoresis tank with buffer.
  • DNA mixed with loading buffer sinks to the bottom of wells; DNA migrates from cathode to anode based on size.

Visualization of DNA Fragments

  • Stain DNA fragments with ethidium bromide (EtBr) to make them fluoresce under UV light; detect above 10 ng of DNA.

Measuring DNA Fragment Size

  • Run DNA size markers alongside unknown fragments on the gel; sizing can be compared visually or by plotting distances against known sizes.

Restriction Mapping

  • Analyze restriction site proportions for specific enzymes in larger molecules derived from agarose gel fragments.

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Description

This quiz covers the fundamentals of restriction enzymes, including their functions and natural roles in protecting cells from foreign DNA. Learn about Type II restriction endonucleases and specific examples such as EcoRI, BamHI, and AluI, as well as the differences between blunt and sticky ends in DNA cutting.

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