Klenow Fragment

It is a large fragment of DNA polymerase I enzyme. It results from the proteolytic cleavage, of DNA polymerase I, caused by the protease subtilisin. Of the 3 main purposes of DNA polymerase I, the Klenow fragment possesses two of them, which are:

·         5’ → 3’ polymerase activity.

·         3’ → 5’ exonuclease activity.

So basically, the Klenow fragment is a DNA polymerase I enzyme, without the 5’ → 3’ exonuclease activity. But having those two properties makes the Klenow fragment have applications such as:

®     The preparation of radioactive DNA probes.

®     DNA sequencing through the Sanger method.

®     Synthesis of second strand of cDNA.

Reference:

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2^nd ed.), 5.40-5.43. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

T4 Polynucleotide Kinase

Ø  Definition:

T4 Polynucleotide Kinase catalyzes the transfer of a phosphate from ATP to the 5´-terminus of polynucleotides or to mononucleotides bearing a 5´-hydroxyl group. The enzyme may be used to phosphorylate RNA, DNA and synthetic oligonucleotides prior to subsequent manipulations such as ligation and cloning.

ATP + 5'-dephospho-DNA {\displaystyle \rightleftharpoons } ↔ ADP + 5'-phospho-DNA        

Ø  Applications:

The T4 polynucleotide kinase is used for labeling the 5'-termini of nucleic acids, and the labeled products can be used int he following ways: - markers for gel-electrophoresis - primers for DNA sequencing - primers for PCR - probes for hybridization - substrates for DNA and RNA ligases - probes for transcrip mapping 5'-phosphorilation of oligonucleotide linkers and DNA or RNA is required before ligation can be performed. using the ³²P-postlabeling assay, DNA modification can be detected.

Ø  Source:

 T4 PNK is purified from E. coli cells expressing a recombinant clone

Ø  Common reaction buffer:

The buffer contains 70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT and pH 7.6 at 25°C

Ø  Structure:

Structure of the 5′‐kinase domain. Two enzyme‐bound sulfates coordinated at the active site tunnel are shown as a stick models. Coordination of one sulfate within the oxyanion hole formed by the P‐loop is depicted by dashed lines.

Ø  References

https://worldwide.promega.com/products/cloning-and-dna-markers/molecular-biology-enzymes-and-reagents/t4-polynucleotide-kinase/?catNum=M4101

https://international.neb.com/products/m0201-t4-polynucleotide-kinase#Product%20Information        

http://emboj.embopress.org/content/21/14/3873.figures-only

https://www.promega.ee/~/media/Files/Resources/Protocols/Technical%20Bulletins/101/T4%20Polynucleotide%20Kinase%20Protocol.pdf

By:

·       Adrian Barnutiu A86084

·       Carlos Rodrigues A85152

·       Catarina Deseyve A85111

·       Gabriel Abreu A84397

·       Matilde Rocha A85740

Enzymes that use DNA as substrate- Nuclease S1

S1 nuclease is a restriction enzyme, specifically an endonuclease (enzyme capable of cleaving the DNA strand at specific sites) derived from the organism Aspergillus oryzae (a fungus).

S1 nuclease is normally present in fungi and plants and is stable at 65°C.

The chemical reaction catalyzed by S1 is an endonucleolytic cleavage that results in 5'-phosphomononucleotides and 5'-phospho-oligonucleotides, requiring Zn²⁺ as the cofactor.

The main function of this enzyme is to remove protrusions in ssDNA (single stranded DNA) and to divide the DNA or RNA (targets preferencially DNA) into oligonucleotides, in order to facilitate the work in molecular biology.

The common reaction buffer is a solution of 10 mM sodium acetate (pH 4.6), 150 mM NaCl, 0.05 mM ZnSO4 and 50% glycerol.

Double-stranded nucleic acids can resist degradation by S1 nuclease except when concentration is extremely high.

Cristia Pires- A94335
Filipa Sampaio-A81577
Marta Machado-A81723
  Marta Mendanha - A82382
 Mónica Fernandes - A84741

Enzymes that use DNA as a substrate - Bam HI

Enzymes that use DNA as a substrate

Bam HI is a restriction endonuclease and that means it cleaves the DNA in a specific sequence of nucleotides. Was isolated from a microorganism, more precisely from Bacillus amyloliquefaciens. This enzyme goes through all DNA molecule and then, when recognizes the sequence GGATCC and cleaves the molecule between the two guanine nucleotides.

Bam HI is more effective at 37ºC and isn’t sensitive to heat inactivation. It is storage at -20ºC in this conditions: (10 mM Tris-HCl; 50 mM KCl; 1 mM DTT; 0.1 mM EDTA; 200 μg/ml BSA; 50% Glycerol; pH 7.4 @ 25°C). Also, Bam HI can cut substrate DNA in 5-15 minutes and can be digested overnight without destroying the DNA.

The principal applications of Bam HI are molecular cloning, restriction site mapping, Genotyping, Restriction fragment length polymorphism (RFLP) and single nucleotide polymorphism (SNP).

The buffer used to incubate the Bam HI is different from company to company, but one of them is: (100 mM NaCl;50 mM Tris-HCl;10 mM MgCl2;100 μg/ml BSA ;(pH 7.9 @ 25°C))

References:

New England Biolabs.inc:

https://international.neb.com/products/r0136-bamhi#Product%20Information_Notes

            ThermoFisher scientific:

https://www.thermofisher.com/order/catalog/product/ER0051

            Protein sequence analysis and classification:

http://www.ebi.ac.uk/interpro/entry/IPR004194

Grupo2.3: Sofia Salgado, Simão Ferreira, Tânia Castro, Tiago Ribeiro, Eduardo Faia

Applied Biology 2^nd year, University of Minho

AluI

Definition (reaction and biologic origin):

AluI is a restriction enzyme (protein that cuts DNA into fragments according to specific nucleotide sequences). It recognizes the sequence AGCT and cuts between the G and C nucleotides.

This enzyme is extracted from E. coli cells whose DNA was modified to carry the gene AluI from Arthrobacter luteus, which is a bacteria commonly found in the soil.

Uses:

   AluI is used in: molecular cloning, restriction site mapping (method used to map an unknown segment of DNA by breaking it into pieces and then identifying the locations of the cuts), genotyping (determines differences in genomes by comparing a DNA sequence to that of another sample or a reference sequence, and it can identifie small variations within populations), restriction fragment length polymorphism (RFLP) and single nucleotide polymorphism (SNP).

Buffer:

The buffer used in reactions with AluI is Tango buffer which is premixed with BSA (Bovin Serum Albumin, a protein derivated from cows that gives stability to the enzymes and binds with contaminants that may be presente in DNA preparations) and ensures the optimum reaction conditions for restriction enzymes.

References:

Unknown author. Date unknown. AluI. Retrieved on November 14, 2018. Available at: https://international.neb.com/products/r0137-alui#Product%20Information

Unknown author. Date unknown. AluI restriction enzyme. Retrieved on November 14, 2018. Available at: https://www.takarabio.com/products/cloning/restriction-enzymes/alui

Unknown author. Date unknown. AluI (10 U/µL). Retrieved on November 15, 2018. Available at: https://www.thermofisher.com/order/catalog/product/ER0011

Unknown author. Date unkown. What is Genotyping? Retrieved on November 15, 2018. Available at:https://www.thermofisher.com/pt/en/home/life-science/pcr/real-time-pcr/real-time-pcr-learning-center/genotyping-analysis-real-time-pcr-information/what-is-genotyping.html

Unknown author. 2014. Restriction mapping. Retrieved on November 15, 2018. Available at: https://www.nature.com/scitable/definition/restriction-mapping-283

Restriction enzymes: Sau3AI

Restriction enzymes, also known as endonucleases, are a type of proteins that cleave the phosphodiester bonds of DNA chains at or near a specific recognition site (the sequence of nucleotides is located by the enzyme itself). These enzymes can be isolated from the bacterial cells that produce them and then used to manipulate fragments of DNA (that may contain genes). For this reason, restriction enzymes are indispensable tools for recombinant DNA technology and genetic engeneering. Their applications include molecular cloning, genotyping, RFLP, SNP, etc.

Sau3AI is an example of a type II restriction enzyme, cutting DNA close or within the nucleotide sequence. In this case, the nucleotide sequence recognized by the enzyme is _5’GATC_3’ , cleaving the DNA chain before the G (guanine) nucleotide. Its optimal reaction temperature is 37ºC and it should be stored at -20ºC to prevent its activity.

                                 Figure 1- Crystal structure of the C-terminal of Sau3AI fragment.

References:

• Thermo Fisher Scientific. (2016). Bsp143I (Sau3AI) (10 U/µL). Consulted on november 16th 2018: https://www.thermofisher.com/order/catalog/product/ER0781

• National Center for Biotechnology Information. (2014). 4PXG: Crystal Structure Of Typeii Restriction Enzyme Sau3ai. Consulted on november 16th 2018: https://www.ncbi.nlm.nih.gov/Structure/pdb/4PXG

• ENCYCLOPÆDIA BRITANNICA. (2018). Restriction enzyme. Consulted on november 16th 2018: https://www.britannica.com/science/restriction-enzyme

• New England Biolabs. Sau3AI. Consulted on november 16th 2018: https://www.neb.com/products/r0169-sau3ai#Product%20Information

Enzymes that use DNA as a substract - DNA Polymerase I

Figure 1 - Functional domains in the Klenow Fragment (left)
and DNA Polymerase I (right).

DNA polymerase I is an enzyme that participates in the DNA replication of prokaryotes. It is the predominant and most abundant polymerizing enzyme found in E.coli. It was the first DNA polymerase to be identified and discovered by Arthur Kornberg, an American biochemist in 1956. He was able to characterize the enzyme initially in E.coli. In E.coli the enzyme is a single polypeptide chain comprised of 928 aminoacids and it has a molecular weight of 109kDa. Nevertheless, the enzyme does not occur only in E.coli but also in many prokaryotes. In prokaryotes it is mostly encoded by polA gene.

Uses:

It has three distinct functions: 3′ to 5′ exonuclease or “proofreading” function because it can remove a nucleotide from the 3’ terminus which was incorrectly inserted in the DNA strand, 5′ to 3′ exonuclease and 5′ to 3′ polymerase.

The second function is the 5’ to 3’ exonuclease. This enzyme has the ability to remove nucleotides from the 5’ terminus. Furthermore, the nucleotides removed can be either of the deoxyribonucleotides or ribonucleotides. The main goal of the 5′ to 3′ exonuclease activity is to remove ribonucleotide primers that are used in DNA replication.

The 5’ to 3’ polymerase is the main activity of the enzyme and it means that it can add nucleotides to the 3’-OH terminus of the previous nucleotide in a DNA template. In order for polymerase to start adding nucleotides it needs a primer (consisted of approximately 10 ribonucleotides) to begin DNA synthesis. DNA pol I adds approximately 15-20 nucleotides per second.

Common reaction buffer:

A common reaction buffer is Tris-HCl (pH 7.2).

References:

ScienceDirect, DNA Polymerases,, viewed in November 13, 2018, https://www.sciencedirect.com/topics/neuroscience/dna-polymerase-i

Worthington Biochemical Corporation, DNA Polymerase I, viewed in November 13, 2018, http://www.worthington-biochem.com/DNAECI/default.html

Biology Online, DNA Polymerase I, viewed in November 13, 2018, https://www.biology-online.org/dictionary/DNA_polymerase_I

Technical information of kit M205A, DNA polymerase I, Promega Corporation.

Evangelia Nikou (e8749) | Hugo Rodrigues (a85946) | Mélanie Pereira (a83980) 

Pedro Gonçalves (a84784) | Ricardo Fernandes (a86254)

University of Minho | School of Sciences | Department of Biology 
Degree in Applied Biology | Genes and Genomes