GTB 204 4th lecture

Once the pure sample of DNA have been prepared, the next step for a molecular biologist is to construct the recombinant DNA molecule. To produce this recombinant molecule : the vector, as well as the DNA that to be cloned/manipulated, must be cut as a specific points and then joined together in a controlled manner. Cutting and joining DNA molecule are two `famous examples of DNA manipulation techniques. As well as being cut and joined, DNA molecule can be shortened, lengthened, copied into RNA or into new DNA molecule, and interestingly it can also be modified by the addition or removal of specific chemical groups.

Common enzymes used in DNA manipulation

Dr. Shaharum Shamsuddin Shaharum@kb.usm.my

These manipulations, all of which can be carried out in the test tube provide the foundation of not only for gene cloning, but also the studies into DNA biochemistry, gene structure and the control of gene expression. Almost all DNA manipulative techniques make use of purified enzymes. Originally, these enzymes exist within the cells and participate in essential process such as DNA replication, transcription, breakdown of unwanted or foreign DNA, repair of mutated DNA and recombination between different DNA molecules. After purification from cells extract, many of these enzymes can be persuaded to carry out their natural reaction, under artificial conditions (or something closely related to them).

The Range of DNA Manipulative Enzymes

These enzyme can be grouped into 5 broad classes depending on the type of reaction they catalyse : 1. Nucleases : Enzymes that cut, shorten or degrade nucleic acid molecules. 2. Ligases : Join nucleic acid molecules together 3. Polymerase : Make copies of molecules 4. Modifying enzymes : Removed or add chemical groups. 5. Topoisomerases : Introduce or remove supercoils from covalently closed circular DNA

Nucleases - Degrade DNA molecule by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand. - There are two kinds of nucleases : 1. Exonucleases : Removed nucleotide one at a time from the end of a DNA molecule (eg. Nuclease BAL31, E. coli Exonuclease III etc) 2. Endonuclease : Break internal phosphodiester bonds within a DNA molecule (eg. S1 Nuclease, Mung Bean Nuclease, DNAse I, RNAse A, Restriction enzyme etc)

An Exonuclease
Cleavage

Hydrogen bond

Phosphodiester bond

nucleotide

Exonuclease remove nt from the end of a DNA molecule

An Endonuclease
Cleavage

RNAses 1. RNAse A : Endoribonuclease that specifically attacks SS RNA and not DNA 2. RNAse H : Endoribonuclease that digest the RNA of an RNA~DNA hybrid

Endonuclease breaks internal phosphodiester bonds

Ligases It repairs single stranded (ss)-break in one of the strands of a double stranded (ds) molecule It also join together individual DNA molecule or the two cohesive ends of the same molecule Mode of action : Catalyzes the formation of a phosphodiester bond between adjacent 3-OH and 5-P termini in DNA

Ligation of complementary sticky ends is much more efficient compare to the ligation of two blunt ends : this is because compatible sticky ends can base pair with one another by hydrogen bonding - forming a relatively stable structure for the enzyme to work on.

Ligating blunt ends

Ligating sticky ends

T4 DNA Ligase

Polymerases The enzymes that synthesis a new strand of DNA complementary to an existing DNA or RNA template Most Polymerases need a template 4 types of DNA Polymerases are used routinely in molecular biology techniques : 1. DNA Polymerase I (usually from. E. coli & T4 phage) :
5--> 3 Polymerase ; 5--> 3 exonuclease & 3--> 5 exonuclease (Enzyme with dual functions - DNA Polymerization & DNA degradation). Commonly used in Nick Translation & Probe preparation, Repairing of DNA fragments, producing a blunt end DNA from a sticky ends DNA

2. Klenow Fragment DNA Polymerase

having 5--> 3 Polymerase & 3--> 5 exonuclease activities No 5-->3 exonuclease activity Can only synthesis a complementary DNA strand on a single stranded template Commonly use in Sanger dideoxy sequencing, synthesis of second strand cDNA in cDNA cloning, Filling in the 3 recessed termini created by digestion of DNA with RE & labeling the termini of DNA fragment - end filling reaction.

3. Reverse Transcriptase (RNA-dependent DNA Polymerase)

Need RNA as a template Having 5-->3 polymerase, 5-->3 riboexonuclease and 3-->5 exoribonuclease activities Commonly used in the synthesis of cDNA for cloning Labeling the termini of DNA fragments with protruding 5 ends (filling reaction).

DNA Modifying enzymes These are enzymes that can modify DNA molecules by addition or removal of specific chemical groups. The most important for this course are : 1. Alkaline phosphatase (AP) : Removes the Phosphate group present at the 5 terminus of a DNA molecule. Uses : To prevent recircularization of plasmid during cloning work. 2. Polynucleotide Kinase : Adding Phosphate groups on to free 5 termini (reverse of AP). 3. Terminal deoxynucleotidyl transferase : Add 1 or more deoxynucleotides onto the 3 terminus of a DNA molecule. Uses : 3 tailing reaction.

4. Taq DNA Polymerase (PCR enzyme)

Only having 5-->3 polymerase activity ONLY, no 3-->5 exonuclease (No proofreading activity !). Widely used in PCR reaction (require specific primers) Latest version of Taq has Proofreading activities with even higher Polymerization capabilities.

4. DNA Methylase (dam & dcm) : Transfer of methyl group to internal A or C residues in the specific sequences to produced methylated duplex DNA.

Classes of Restriction Endonucleases

Class I

Description
Recognize some specific sequence like all other Res, but they are not particularly useful in gene manipulation since their cleavage site is non-specific. They have methylase activity They are Mg2+ dependent with specific recognition site. Very useful for DNA manipulation work. Contain nuclease & methylase activity. Recognition site are not symmetrical

II III

Restriction Enzymes (Class II RE) These are enzymes for cutting DNA in a very PRECISE and REPRODUCEABLE fashion during molecular cloning work. They cut both strands of DS DNA within a (normally symmetrical) recognition sequence. Hydrolyze sugar phosphate backbone to give a 5-phosphate on one side and 3-OH on the other. Yield blunt or sticky ends (5 or 3 overhang). The discovery of these enzymes which led to Nobel Prize for Arber, W., Smith, H and Nathans, D. in 1978, was one of the key breakthroughs in the development of genetic engineering.

Some Important Terminologies using REs Blunt Ends Sticky ends

Isoschizomer : Different REs (from different sources) that can recognize the same restriction site - ther are isoschizomers. Palindromic sequence : `Read alike, backward and forward eg. Restriction site for Eco R1 : - GAA - CT T TTC AAG -

Nomenclature of REs Proposed by Smith & Nathans (1973) : Species name of the organism that produce the enzyme : Escherichia coli = Eco ; Haemophilus influenza = Hin Strain will be written as subsript : Eg. Ecok

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If the strain has different Restriction or Modification systems, these will be identified by Roman Numerals : Eg. H. Influenzae strain Rd will be = R. Hind I ; R. HindII. ; R. HindIII

Spaciba !