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Modifying Enzymes
Function, enzymatic activity, applications of modifying enzymes
| Term | Definition |
|---|---|
| Thermosensitive Alkaline Phosphatase - Function | Removes 5´- and 3'- phosphate groups from DNA, RNA, nucleotides and proteins |
| Thermosensitive Alkaline Phosphatase - Applications | Preventing the recircularization and religation of linearized cloning vector DNA during ligation |
| Thermosensitive Alkaline Phosphatase - Temp Sensitivity | Works efficiently at 37oC and deactivated at 75oC |
| T4 Polynucleotide Kinase (T4 PNK) - Function | Cleaves a g-phosphate group (P) from ATP and transfer to the 5’ end of DNA/RNA (Replacing the –OH on the sugar group) |
| T4 Polynucleotide Kinase (T4 PNK) - Applications | Phosphorylate RNA, DNA and synthetic oligonucleotides prior to subsequent manipulations such as ligation and cloning |
| T4 DNA Ligase - Function | Catalyzes phosphodiester bond between close 5'-phosphate and 3'-hydroxyl termini in DS DNA/RNA. Repairs SS nicks in DS DNA, RNA, or DNA/RNA hybrids, joins DNA fragments with cohesive/blunt termini (no activity on SS nucleic acids) Needs ATP as cofactor |
| T4 DNA Ligase - What does all ligation need? | All ligation needs 5’-Phosphate group. Restriction enzyme digest/kinase treatment can generate 5’-Phosphate. Primers ordered through commercial sources usually do not have 5’-phosphate. One will need to treat PCR amplicons with kinase prior to ligation |
| DNA Polymerase I (Pol 1) - Function | 3 catalytic sites: -5'→3' (forward) DNA-dependent DNA polymerase activity, requires 3' primer site and template strand -3'→5' (reverse) exonuclease activity for proofreading -5'→3' (forward) exonuclease activity for nick translation during DNA repair |
| DNA Polymerase I (Pol 1) - Applications | Physiological function: supporting repair of damaged DNA, helps connect Okazaki fragments by deleting RNA primers & replacing with DNA. DNA replication & proof-reading (fill DNA gaps arising in DNA replication, repair and recombination) Nick translation |
| DNA Polymerase I (Pol 1) - Origin | Discovered by Arthur Kornberg in 1956 and was the first known DNA polymerase (also the first known of any kind of polymerase). It was initially characterized in E. coli and is ubiquitous in prokaryotes. |
| Klenow Fragment - Origin | Klenow fragments are produced by digesting DNA polymerase 1 with a protease called subtilisin |
| Klenow Fragment - Function | Like Pol 1, Klenow fragment have 5’to 3′ DNA-dependent DNA polymerase activity and 3’to 5′ exonuclease activity that mediates proofreading |
| Klenow Fragment - Applications | Synthesis of DS DNA from SS templates. Filling in receded 3' ends to blunt 5'-overhangs. Digesting protruding 3' overhang. Prepping radioactive DNA probes -Random-primed DNA labeling -Site-specific mutagenesis of DNA with synthetic oligos |
| Klenow Fragment - The first enzyme to be used for which method? | The Klenow fragment was the original enzyme used during the first protocols of PCR amplification, before being replaced by thermostable enzymes such as Taq polymerase. |
| exo-Klenow Fragment - Function | Large fragment of DNA polymerase I. Only has 5′ to 3′ polymerase activity & lacks the 3'→5' and 5'→3' exonuclease activities of DNA Pol I. The 3'→5' exonuclease activity of the enzyme is eliminated by mutations in the 3'→5'-exonuclease active site |
| exo-Klenow Fragment - Applications | Fluorescent labeling rxns for microarray. dA and dT tailing (key step when ligating DNA adapters to DNA fragments). Prepping DNA libraries for NGS -Incorporating modified nucleotides (e.g., Cy3-, Cy5-, fluorescein-, biotin-labeled nucleotides, etc) |
| T4 DNA Polymerase - Functions | Template-dependent DNA polymerase that catalyzes 5'-3' synthesis from primed single-stranded DNA. Has a 3'-5' exonuclease activity, but lacks 5'-3' exonuclease activity |
| T4 DNA Polymerase - Applications | Blunting DNA ends: fill in 5'-overhangs/removal of 3'-overhangs. Blunting PCR products with 3'-dA overhangs. Synthesize labeled DNA probes via replacement rxn -Oligo-directed site-specific mutagenesis -Ligation-independent cloning of PCR products |
| T4 DNA Polymerase - How does it compare to other mod enzymes? | Stronger 3'-5' exonuclease activity on single-stranded than on double-stranded DNA and greater (more than 200 times) than DNA polymerase I, E. coli,and Klenow fragment |
| Uracil-DNA Glycosylase (UDG, UNG) - Function | Removes uracil residues from the sugar moiety of SS/DS DNA without destroying the phosphodiester backbone, preventing its use as a hybridization target or as a template for DNA polymerases (UDG will not remove uracil from RNA) |
| Uracil-DNA Glycosylase (UDG, UNG) - Applications | Helps to eliminate carryover contamination in PCR, resulting in fewer false positive results for cloning PCR fragments |
| DNase I (RNase-free) - Function | DNA specific Endonuclease that degradesDS and SS DNA. Products are short oligos with 5'-phosphate and 3'-OH |
| DNase I (RNase-free) - Applications | Removal of contaminating genomic DNA from RNA samples. Degradation of DNA templates in transcription reactions. DNase I footprinting. Nick Translation |
| T7 RNA Polymerase - Function | DNA-dependent RNA polymerase with strict specificity for its respective DS promoters. Catalyze 5'→3' synthesis of RNA on SS/DS DNA downstream from its promoter Synthesizes RNA faster than E. coli RNA polymerase & terminates transcription less frequently |
| T7 RNA Polymerase - Applications | Synthesis of un/labeled RNA that can be used for hybridization, in vitro RNA translation, as aRNA/siRNA/substrate in RNase protection assays, template for genomic DNA sequencing, & studies of RNA 2nd structure, RNA-protein interactions and RNA splicing |
| Endonuclease IV (Endo IV) - Function | Cleaves middle DNA phosphodiester backbone at AP sites (missing base) via hydrolysis leaving 1 ncld gap with 3'-OH and 5' deoxyribose phosphate (dRP) termini. 3'-diesterase activity: removes 3' P, 3'-α, β-unsaturated aldehyde and other 3' blocking groups |
| Endonuclease IV (Endo IV) - Applications | Studies of DNA damage and repair. Single cell electrophoresis (comet assay). Antitumor drug research. DNA structure research • SNP analysis |
| RiboLock / RNaseOUT RNase Inhibitor - Function | Ribonuclease inhibitors protects RNA from degradation and contamination. RNase Inhibitor inhibits the activity of RNases A, B and C by binding in a 1:1 ratio |
| RiboLock / RNaseOUT RNase Inhibitor - Applications | Vitro Translation, Vitro Transcription, qRT-PCR, and cDNA Synthesis |
| RiboLock / RNaseOUT RNase Inhibitor - Which is Invitrogen brand? | RiboLock RNase Inhibitor = Thermo Scientific. RNaseOUT RNase Inhibitor = Invitrogen |
| Endonuclease V, T. maritima (Endo V) - Function | 3'-endo for DNA repair: removes deaminated bases from damaged DNA (uracil, hypoxanthine, xanthine). Acts on abasic/urea sites, base pair mismatches, flap/pseudo Y structures & tiny insertions/deletions inDNA Cleaves 2nd phosphodiester bond 3' to lesion |
| Endonuclease V, T. maritima (Endo V) - Applications | High-throughput methods for mutation research. Studies in mutagenesis and DNA repair. Mismatch cleavage. Genotyping |
| Single-Stranded DNA Binding Protein (SSB) - Function | Bind with high affinity to single-stranded DNA (does not bind well to double-stranded DNA). After binding single-stranded DNA, SSB destabilizes helical duplexes, allowing DNA polymerases to access their substrate more easily |
| Single-Stranded DNA Binding Protein (SSB) - Applications | Enhance DNA polymerase activity. Fluorescence polarization assays. Enables longer reads in pyrosequencing (SNP analysis) Stop pausing when sequencing regions of SS/DS DNA with much 2nd structure Improve RE digestion Site-directed mutagenesis w/ RecA |
| Exonuclease I (ExoI) - Function | Degrades ssDNA in a 3'→5' direction. It releases deoxyribonucleoside 5'-monophosphates in a stepwise manner, leaving 5'-terminal dinucleotides intact. It does not cleave DNA strands with terminal 3'-OH groups blocked by phosphoryl or acetyl groups. |
| Exonuclease I (ExoI) - Applications | Primer removal from PCR mixtures --> prior to PCR product sequencing |
| Exonuclease VII (Exo VII) - Function | Cleaves linear single-stranded DNA in both the 3' to 5' and 5' to 3' directions. Preferred substrate is linear single-stranded DNA |
| Exonuclease VII (Exo VII) - Applications | Removal of primers with or without 3' or 5' terminal phosphorothioate bonds. Mapping positions of introns in genomic DNA. Removal of single-stranded DNA, leaving behind the double-stranded DNA in a sample |
| Pyrophosphatase, Inorganic - Function | Catalyzes the hydrolysis of inorganic pyrophosphate (an inhibitor to nucleic acid synthesis) into two orthophosphates |
| Pyrophosphatase, Inorganic - Applications | High yield RNA synthesis by in vitro transcription. DNA polymerization reactions: preventing accumulation of pyrophosphate. Removal of contaminant PPi in reagents used for SNP genotyping by methods based on the detection of pyrophosphate |
| RNase H - Function | Specifically degrades the RNA strand in RNA-DNA hybrids. Will not degrade DNA or unhybridized RNA. Integral part of most RNA amplification and NASBA protocols |
| RNase H - Applications | Remove mRNA before 2nd strand cDNA synthesis. RT-PCR & qRT-PCR: remove RNA after 1st strand cDNA synthesis Site-specific cleavage of RNA (remove poly(A) tail when complementary DNA oligo is hybridized) Studies of invitro polyadenylation rxn products |
Created by:
zymathon
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