L9 DNA&RNA Modifying Enzymes

一、Polynucleotide Kinase & Alkaline Phosphatase

T4 polynucleotide Kinase

Origin: T4 phage

Feature:

1. Catalyzes the transfer of the gamma-phosphate from ATP to the 5’-OH group of singleand double-stranded DNAs and RNAs.
2. The enzyme is also a 3’-phosphatase under appropriate condition

Usage:

1. 5’ phosphorylation of DNA/RNA for subsequent ligation
2. End labeling DNA or RNA for probes and DNA sequencing
3. Phosphorylation of PCR primers
4. Removal of 3’ phosphoryl groups

Alkaline Phosphatase

Origin: Calf or Shrimp

Feature:

1. Alkaline Phosphatase catalyzes the release of 5’and 3’-phosphate groups from DNA, RNA, and nucleotides.
2. This enzyme also removes phosphate groups from proteins
3. CIAP (Calf intestinal AP) & SAP (Shrimp AP).
4. Phosphatase needs to be inactivated before the DNA is used for ligation.

Usage:

1. removes 3´ and 5´ phosphate group from DNA, RNA and dNTP
2. To prevent self-ligation

二、Terminal Deoxynucleotidyl Transferase & Poly(A) Polymerase

Terminal Deoxynucleotidyl Transferase

Origin: Calf thymus

Feature:

1. TdT is a template-independent DNA polymerase that catalyzes the repetitive addition of deoxyribonucleotides to the 3’-OH of oligodeoxyribonucleotides and single-stranded and double-stranded DNA

**Usage: **

1. To create single-stranded overhang of dsDNA – for gene cloning
2. DNA 3’end labling.
   • Creats a sticky end
   • 

Poly(A) Polymerase

Origin: E.coli or Yeast

Feature:

1. catalyzes the addition of adenosine to the 3’ end (-OH) of RNA in a sequence-independent fashion

**Usage: **

1. Labeling RNA 3’ end; facilitate reverse transcription, cloning & sequencing.

三、Nucleases – DNase I, Exonuclease I, RNase A, RNase I, RNase, H, MNase

Nucleases – DNase I

Origin: E.coli or Yeast

Feature:

1. A nonspecific endonuclease that degrades dsDNA, ssDNA and DNA in RNA:DNA hybrid.
2. It functions by hydrolyzing phosphodiester linkages, producing mono and oligonucleotides with a 5’-phosphate and a 3’-hydroxyl group

Usage:

1. Preparation of DNA-free RNA.
2. DNA labeling by nick-translation in conjunction with DNA Polymerase I
3. Studies of DNA-protein interactions by DNase I footprinting

DNase I foot-printing to study DNA-protein interaction:

Exonuclease I (E. coli)

Origin: E.coli

Feature:

1. 3’ to 5’ exonuclease of ssDNA
2. Catalyzes the removal of nucleotides from linear single stranded DNA in the 3’ to 5’ direction

Usage:

1. Remove excessive primers from PCR product
   • Exo-SAP treatment
     • The exonuclease I removes leftover primers, while the Shrimp Alkaline Phosphatase removes any remaining dNTPs

RNase A

Degrades single-stranded RNA at C and U residues.

Cleaves the phosphodiester bond between the 5’-ribose of a nucleotide and the phosphate group attached to the 3’-ribose of an adjacent pyrimidine nucleotide (C or U).

Leave a 3’ end phosphate group after clevage.

Resistant to heat, easy to refold – the enzyme that we should worry about in RNA extraction.

RNase I

RNase I degrades single-stranded RNA to nucleoside 3´ monophosphates via 2´,3´ cyclic monophosphate intermediates by cleaving between all dinucleotide pairs

The enzyme is completely inactivated by heating at 70°C for 15 minutes.

A more controllable choice to digest RNA than RNase A

RNase H

specifically degrades the RNA strand of an RNA-DNA hybrid to produce 5’ phosphate terminated oligoribonucleotides and single-stranded DNA

Application:

• remove RNA from cDNA

Micrococcal nuclease (MNase)

Exhibits exoand endo-5’-phosphodiesterase activities against DNA and RNA. ds/ss DNA/RNA.

• cut them all

Preference for ATor AU-rich regions.

Strictly dependent on calcium for digestion of RNA and DNA substrates. Precisely inactive the enzyme by adding EGTA

Application: remove RNA from cDNA

• Remove nucleic acids from solution
• Release nucleosome from chromatin

Proteinase K

Cloned from Engyodontium album, is a nonspecific serine protease that is useful for general digestion of proteins.

Optimal activity between 6.5 and 9.5

Active under denaturing conditions, SDS or urea

Active under high temperature, 55-65 degree

Application:

• remove proteins from solution/cell extract (e.g. DNA or RNA extraction)

Summary

1. Polynucleotide Kinase (Both a Kinase and phosphatase)
2. Alkaline Phosphatase (You need to remove it before ligation)
3. Terminal Deoxynucleotidyl Transferase (For add a stretch of A on DNA)
4. Poly(A) Polymerase (For add a stretch of A on RNA).
5. Nucleases – DNase I, Exonuclease I, RNase A, RNase I, RNase H, MNase (They all cut nucleic acids with different features，think of a scenario that you may use it).