Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Using Restriction Enzymes to Make Recombinant DNA Bacteria and Archaea have evolved restriction enzymes that cut invading DNA molecules at a limited number of specific DNA sequences, called restriction sites. We have now discovered over 3,000 restriction enzymes.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Using Restriction Enzymes to Make Recombinant DNA The restriction sites are palindromes and a restriction enzyme will usually make many cuts in a DNA molecule yielding a set of restriction fragments. The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that can bond with complementary “sticky ends” of other fragments. DNA ligase will then seal the bonds back up between the restriction fragments.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Restriction Endonucleases Type II Restriction Enzymes At the restriction site, one side of the DNA molecule is a palindrome to the other. 5` CTTAAG 3` 3` GAATTC 5`
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Restriction Endonucleases Ligation with DNA Ligase to finish the job. DNA ligase joins DNA molecules or fragments by creating a phosphodiester bond between an adjacent 3’ OH and a 5’ PO 4 -.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Restriction Endonucleases Examples of Type II Restriction Endonucleases Blunt ends Cohesive “sticky” ends Where do restriction enzymes get their names?
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Restriction Endonucleases Examples of Type II Restriction Endonucleases Where do restriction enzymes get their names? Escherichia coli An E. coli strain that carries the cloned Hpa I gene from Haemophilus parainfluenzae. Haemophilus influenzae An E. coli strain that carries the cloned Pst I gene from Providencia stuartii.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Restriction Endonucleases The Restriction-Modification System of Bacteria Bacteria can protect themselves from a phage virus by methylating the Adenines at the recognition site. Bacteriophage DNABacterial DNA
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Applications Genetically Modified Organisms Gene that produces a unique trait (ex: herbicide resistance. Soybean DNA with a nonessential gene. Soybean plant now has resistance to the herbicide.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Forensic Evidence DNA “fingerprints” obtained by analysis of tissue or body fluids found at crime scenes can provide definitive evidence that a suspect is guilty or not. DNA fingerprinting can also be used in establishing paternity.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings A VERY simplified look at the process: 1.DNA discovered at crime scene. 2.Amplify DNA with Polymerase Chain Reaction. 3.Cut DNA with known restriction enzyme (e.g. EcoRI) to produce Restriction Fragment Length Polymorphisms (RFLP’s). 4.Analyze RFLP’s with gel electrophoresis.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The PCR procedure:
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Restriction fragment analysis can rapidly provide useful comparative information about DNA sequences. Gel electrophoresis separates DNA restriction fragments of different lengths.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel: DNA segments with EcoRI restriction sequences in the same place will produce RFLP’s of the same size and thus the same banding pattern.
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings