BTT 306 Techniques in Biotechnology Topic 2: Enzymes used in cloning Lim Theam Soon INFORMM, USM Enzymes: An enzyme is a biological catalyst and is almost always a protein. It speeds up the rate of a specific chemical reaction in the cell. The enzyme is not destroyed during the reaction and is used over and over. DNA manipulative enzymes can be grouped into four broad classes, depending on the type of reaction that they catalyze: Nucleases - enzymes that cut, shorten, or degrade nucleic acid molecules • Ligases - join nucleic acid molecules together • Modifying enzymes - remove or add chemical groups https://www.genome.gov/genetics-glossary/Enzyme How to cut DNA? Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next in a DNA strand. There are two different kinds of nuclease: Exonucleases remove nucleotides one at a time from the end of a DNA molecule. Endonucleases are able to break internal phosphodiester bonds within a DNA molecule. The main distinction between different exonucleases lies in the number of strands that are degraded when a double-stranded molecule is attacked. Restriction Endonucleases A restriction enzyme is a DNA-cutting enzyme that recognizes a specific target sequence and cuts DNA into two pieces at or near that site. Many restriction enzymes produce cut ends with short, single-stranded overhangs. Restriction enzymes are found in bacteria (and other prokaryotes). They recognize and bind to specific sequences of DNA, called restriction sites. Each restriction enzyme recognizes just one or a few restriction sites. When it finds its target sequence, a restriction enzyme will make a double-stranded cut in the DNA molecule. Typically, the cut is at or near the restriction site and occurs in a tidy, predictable pattern. https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/restriction-enzymes-dna-ligase Different cuts There are two types of cuts that can be generated depending on the enzyme. Blunt End Digestion https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/restriction-enzymes-dna-ligase Overhang Digestion Types of restriction enzymes Restriction enzymes are traditionally classified into four types on the basis of subunit composition, cleavage position, sequence specificity and cofactor requirements. Type I Enzymes Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. Type III Enzymes Type III enzymes are also large combination restriction-and-modification enzymes. They cleave outside of their recognition sequences and require two such sequences in opposite orientations within the same DNA molecule to accomplish cleavage; they rarely give complete digests. Type II Enzymes Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and they are the predominant class used in the laboratory for routine DNA analysis and gene cloning. Type IIS enzymes cleave outside of their recognition sequence to one side. These enzymes are intermediate in size, 400650 amino acids in length, and they recognize sequences that are continuous and asymmetric. Type IV Enzymes Type IV enzymes recognize modified, typically methylated DNA. https://international.neb.com/products/restriction-endonucleases/restriction-endonucleases/types-of-restriction-endonucleases Type IIG enzymes are large, combination restrictionand-modification enzymes, 850-1250 amino acids in length, in which the two enzymatic activities reside in the same protein chain. These enzymes cleave outside of their recognition sequences and can be classified as those that recognize continuous sequences and cleave on just one side; and those that recognize discontinuous sequences and cleave on both sides releasing a small fragment containing the recognition sequence. How to stick DNA? In the cell the function of DNA ligase is to repair single-stranded breaks (“discontinuities”) that arise in double-stranded DNA molecules during, for example, DNA replication. DNA ligase covalently joins the phosphate backbone of DNA with blunt or compatible cohesiveends and it’s natural role is in repairing double strand breaks in DNA molecules. In molecular biology it is commonly used for the insertion of restriction enzyme-generated DNA fragments into vector backbones. Commercial ligases are supplied with a reaction buffer containing ATP and Mg2+, which are both essential for ligase activity. How Ligase works? DNA ligase catalyzes the joining of the 3′-OH to the 5′-phosphate via a two step mechanism:1.The AMP nucleotide, which is attached to a lysine residue in the enzyme’s active site, is transferred to the 5′-phosphate. 2.The AMP-phosphate bond is attacked by the 3′-OH, forming the covalent bond and releasing AMP. To allow the enzyme to carry out further reactions the AMP in the enzyme’s active site must be replenished by ATP. Other modifying enzymes The enzymes work to introduce modification either by adding or removing certain chemical groups. • Alkaline phosphatase (from E. coli, calf intestinal tissue, or arctic shrimp), which removes the phosphate group present at the 5′ terminus of a DNA molecule. • Polynucleotide kinase (from E. coli infected with T4 phage), which has the reverse effect to alkaline phosphatase, adding phosphate groups onto free 5′ termini. • Terminal deoxynucleotidyl transferase (from calf thymus tissue), which adds one or more deoxyribonucleotides onto the 3′ terminus of a DNA molecule. https://en.wikipedia.org/wiki/Molecular_cloning https://en.wikipedia.org/wiki/Molecular_cloning Subcloning https://worldwide.promega.com/resources/guides/nucleic-acid-analysis/subcloning/#subcloning-strategies-c31cbe67-c0f3-4a0d-b871-de1c671e75f2 Subcloning Strategy: Common Restriction Sites https://worldwide.promega.com/resources/guides/nucleic-acid-analysis/subcloning/#subcloning-strategies-c31cbe67-c0f3-4a0d-b871-de1c671e75f2 Subcloning Strategy: Common Restriction Sites with Partial Digests https://worldwide.promega.com/resources/guides/nucleic-acid-analysis/subcloning/#subcloning-strategies-c31cbe67-c0f3-4a0d-b871-de1c671e75f2 Subcloning Strategy: Moving Inserts with Compatible Restriction Sites https://worldwide.promega.com/resources/guides/nucleic-acid-analysis/subcloning/#subcloning-strategies-c31cbe67-c0f3-4a0d-b871-de1c671e75f2 Subcloning Strategy: Moving Inserts with Only One Common Site https://worldwide.promega.com/resources/guides/nucleic-acid-analysis/subcloning/#subcloning-strategies-c31cbe67-c0f3-4a0d-b871-de1c671e75f2 Subcloning Strategy: Blunt-End Method Concern: Orientation cannot be controlled https://worldwide.promega.com/resources/guides/nucleic-acid-analysis/subcloning/#subcloning-strategies-c31cbe67-c0f3-4a0d-b871-de1c671e75f2 Vector to insert ratio for cloning Vector: Insert molar ratios between 1:1 and 1:10 are optimal for single insertions (up to 1:20 for short adaptors). Insert: vector molar ratio should be 6:1 to promote multiple inserts. Example: How much 0.5kb insert DNA should be added to a ligation in which 100ng of 3kb vector will be used? The desired vector:insert ratio will be 1:2. https://worldwide.promega.com/resources/guides/nucleic-acid-analysis/subcloning/#subcloning-strategies-c31cbe67-c0f3-4a0d-b871-de1c671e75f2 Application of alkaline phosphatase to improve cloning experiments Alkaline phosphatase • Alkaline Phosphatase, Calf Intestinal (CIP) nonspecifically catalyzes the dephosphorylation of 5´ and 3´ ends of DNA and RNA phosphomonoesters. • Also, CIP hydrolyses ribo-, as well as deoxyribonucleoside triphosphates (NTPs and dNTPs). • CIP is useful in many molecular biology applications such as the removal of phosphorylated ends of DNA and RNA for subsequent use in cloning or end-labeling of probes. • In cloning, dephosphorylation prevents religation of linearized plasmid DNA. The enzyme acts on 5´ protruding, 5´ recessed and blunt ends. • CIP may also be used to degrade unincorporated dNTPs in PCR reactions to prepare templates for DNA sequencing or SNP analysis. https://biology.stackexchange.com/questions/97944/alkaline-phosphatase-and-ligase-protocol-for-cloning https://international.neb.com/products/m0290-alkaline-phosphatase-calf-intestinal-cip#Product%20Information Lambda exonuclease DNA specific exonuclease Catalyzes the removal of nucleotides from linear or nicked double-stranded DNA in the 5' to 3' direction https://international.neb.com/products/m0262-lambda-exonuclease#Product%20Information Highly processive degradation of doublestranded DNA from the 5' end Preferred substrate is 5'phosphorylated doublestranded DNA although non-phosphorylated substrates are degraded at a greatly reduced rate Conversion of linear double-stranded DNA to single-stranded DNA via preferred activity on 5'phosphorylated ends DNA Polymerase I, Large (Klenow) Fragment DNA Polymerase I, Large (Klenow) fragment was originally derived as a proteolytic product of E.coli DNA polymerase that retains polymerase and 3’ —> 5’ exonuclease activity •Removal of 3’ overhangs or fill-in of 5’ overhangs to form blunt ends •Lacks 5’ —> 3’ exonuclease activity •Generates probes using random primers •Second strand cDNA synthesis https://international.neb.com/products/m0210-dna-polymerase-i-large-klenow-fragment#Product%20Information https://www.abmgood.com/dna-polymerase-i-large-klenow-fragment-e013-vin.html Gene assembly / mutation / swapping https://doi.org/10.2144/000113964 Other Cloning Methods Biobricks Biobricks is a trademark term for man-made DNA sequences encoding elementary modules that may be combined to produce more complex synthetic biological systems. The long-term goal of the Biobricks Foundation is to offer an open-source library of standardized genetic components. Gibson Assembly Gibson assembly is a molecular cloning method which allows for the joining of multiple DNA fragments in a single, isothermal reaction. It is named after its creator, Daniel G. Gibson, who was the chief technology officer and co-founder of Codex DNA. Additional reading topics: • Other modifying enzymes • Cloning methods
