Restriction Enzymes Lecture PDF

Restriction Digestion Analysis BCH405/BIOT401: Bioinformatics Lecture: Restriction Digestion Analysis By Adeena Tahir  Learning Objectives Enzymes Restriction Enzymes Discovery of Enzymes Importance of Restriction Enzymes Typ...

Restriction Digestion Analysis BCH405/BIOT401: Bioinformatics Lecture: Restriction Digestion Analysis By Adeena Tahir  Learning Objectives Enzymes Restriction Enzymes Discovery of Enzymes Importance of Restriction Enzymes Types of Restriction Enzymes Restriction Sites Nomenclature of Restriction Enzymes Summary  Enzymes ▪ Enzymes are biological catalysts – found in all cells. ▪ Enzymes have unique chemical structures that mean they only act on specific substrates ▪ For a particular enzyme to work optimally, it must be under specific conditions (temperature, pH), otherwise its unique chemical structure will be disrupted. ▪ Enzymes are critical for a range of cellular processes including digestion, DNA replication, protein synthesis. What are restriction enzymes? Molecular scissors that cut double stranded DNA molecules at specific points. Found naturally in a wide variety of prokaryotes An important tool for manipulating DNA. ▪ These enzymes were discovered in bacteria. ▪ They help the bacteria destroy viral DNA. (bacteria get viruses too!) ▪ They cut between specific bases (letters) of the double stranded DNA molecule ▪ 3,000 enzymes have been identified, around 200 have unique properties, many are purified and available commercially  Researchers rely on restriction enzymes to assist with many processes in laboratories around the world: Restriction Enzymes - 1. Making recombinant DNA and appraising success For research, medicine and agriculture Purposes 2. DNA profile analysis For disease diagnosis, paternity/family relationship testing, and forensics  A recognition sequence is different from a recognition site A recognition sequence is a DNA sequence to which a structural motif of a DNA-binding domain exhibits binding specificity. Recognition sequences are palindromes. A recognition site is specified by the position of the site. For example, there are two PstI recognition sites in the following DNA sequence fragment, starting at base 9 and 31 respectively. A recognition sequence is a specific sequence, usually very short (less than 10 bases).  Discovery of Restriction Endonuclease Arbor and Dussoix in 1962 discovered that certain bacteria contain Endonucleases which have the ability to cleave DNA. In 1970 Smith and colleagues purified and characterized the cleavage site of a Restriction Enzyme in Hemophilus influenzae. Werner Arbor, Hamilton Smith and Daniel Nathans shared the 1978 Nobel prize for Medicine and Physiology for their discovery of Restriction Enzymes.  Biological Role Most bacteria use Restriction Enzymes as a defense against bacteriophages. Restriction enzymes prevent the replication of the phage by cleaving its DNA at specific sites. The host DNA is protected by Methylases which add methyl groups to adenine or cytosine bases within the recognition site thereby modifying the site and protecting the DNA. Types of Restriction Enzymes Types Of Restriction Enzymes Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IV) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence. All types of enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific fragments with terminal 5'-phosphates.  Restriction sites DNA sites of four to eight nucleotides Palindrome sequences Specific restriction enzymes cut at specific DNA sequences. For example: EcoRI is an enzyme that cuts at the following sequence: GAATTC EcoRI was discovered in E. coli bacteria. The resulting pieces of DNA are called “restriction fragments.”  Many restriction enzymes… HindIII was discovered in H. influenza PstI was discovered in P. stuartii EcoRV was discovered in E. coli Protruding ends are also called “sticky” ends. Why might these be useful?  Restriction fragments can be blunt ended or sticky ended 5’ G A A T T C 3’ 5’ G A T A T C 3’ 3’ C T T A A G 5’ 3’ C T A T A G 5’ Sticky Ends Blunt Ends Sticky ends or blunt ends can be used to join DNA fragments. Sticky ends are more cohesive compared to blunt ends. Not all restriction endonucleases cut symmetrically and leave blunt ends. Many endonucleases cleave the DNA backbones in positions that are not directly opposite to each other or can make staggered ends which produce single stranded “sticky ends”. DNA from different sources can be spliced easily because of these sticky end overhangs. Restriction fragments: sticky ends Restriction fragments: blunt ends Restriction Enzymes & their Recognition sequences  Nomenclature of REs Smith and Nathans (1973): ▪ A three-letter abbreviation (Host organism) ▪ First letter of the genus name. ▪ Two letters of the species name ▪ Abbreviation is always in italics. ▪ Same Host & different enzymes ▪ Identify by roman numerals. Nomenclature of REs  Some Important terms Isoschizomers Restriction enzymes that recognize the same DNA sequence. The cut sites may or may not be identical. Neoschizomers Enzymes that recognize the same DNA sequence but cut at different bases relative to that sequence. These enzymes are a subset of the isoschizomers. Eg:SmaI and XmaI  The Frequency of Restriction Endonuclease Cut Sites The frequency depends : Base composition Length of the recognition sequence  25 DNA and its bonds DNA molecules are macromolecules that hold the genetic information of living organisms. The covalent bond joining adjacent nucleotides in DNA is called a phosphodiester bond. The phosphodiester bonds between nucleotides in DNA molecules are very stable unless they are physically stretched or exposed to enzymes called nucleases.  26 Endo- and Exonucleases Nucleases are enzymes capable of breaking (hydrolyzing) phosphodiester bonds in DNA molecules. Classified into two major groups: 1. Exonucleases: If the enzyme digest nucleotides from the ends of the DNA molecules. 2. Endonucleases: If the enzyme digest nucleotides in the interior of a DNA molecule. Restriction enzyme: A protein that recognizes a particular sequence of DNA and cuts the DNA at that site (the restriction site)  27 Digestion Procedure  28 Steps of DNA Restriction Analysis Step 1 Extraction and purification of DNA  29 Step 2 Digestion Thousands of with restriction fragments Restriction of all different sizes Enzymes  30 Step 3 Gel Electrophoresis  31 Step 4 Southern Blot and hybridization with radioactive probe  32 Step 5 Analysis of the fragment distribution.  33 Restriction Mapping/Digest Analysis Aim: the digested fragments must be separated and identified. Fragments are separated by agarose gel electrophoresis. [Agarose is a large polysaccharide]. Gel electrophoresis: your DNA will move through spaces in agarose against a current DNA has a negative charge and will migrate towards the cathode Example of Restriction Map  36 Restriction Digest Analysis  37 DNA Restriction Analysis This process makes use of special proteins called restriction enzymes and sections of the chromosome called tandem repeats. All humans have the same type of repeats but there is tremendous variation in the number of repeats that each of us has  Summary What are Restriction Enzymes? Discovery of Enzymes Importance of Restriction Enzymes Types of Restriction Enzymes Nomenclature of Restriction Enzymes Conclusion  Take Home Message Abu Musa reported that the Messenger of Allah (S.A.W.W) said: “Allah gives respite to the wrongdoer, then when HE seizes him, HE doesn’t let him go.” (Sunnan Ibn e Maja: 4018)  Thankyou...! Remember to revise your lecture and give Feedback [email protected]