Оберемок В.В. Справочник по ДНК-модифицирующим энзимам и связанным с ними методам молекулярной генетики (на русском и английском языках)

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EXONUCLEASES

Exonuclease I (Exo I)

Exonuclease I (ExoI) degrades single-stranded DNA in

3`→5` direction, releasing deoxyribonucleoside 5`-monophosphates

in a stepwise manner and leaving 5`-terminal dinucleotides intact.

Features:

1) it does not cleave DNA strands with terminal 3`-OH groups

blocked by phosphoryl and acetyl groups;

2) active in buffers for PCR;

3) is not suitable for removing 3`-overhangs of dsDNA.

Applications:

1) primer removal from PCR mixtures;

2) removal of single stranded DNA containing a 3`-hydroxyl terminus

from nucleic acid mixtures;

3) assay for the presence of single-stranded DNA with a 3`-hydroxyl

terminus.

10X Reaction buffer

670 mM glycine-KOH (pH 9.5 at 25

C), 67 mM MgCl

, 10 mM DTT

Inhibition and inactivation

Inhibitors: 20 %(w/v) PEG 8000; inactivated by heating at 80

C for 15 min.

Fig. 8. Exonuclease I activity

Exonuclease III

Exonuclease III (Exo III) exhibits four different catalytic

activities:

1) 3`→5` exodeoxyribonuclease activity specific for double-

stranded DNA: Exo III degrades dsDNA from blunt ends, 5`-

overhangs or nicks, releasing 5`-mononucleotides from the

3`-ends of DNA strands and producing stretches of single

stranded DNA. It si not active on 3`-overhang ends of DNA

that are at least four bases long and do not carry a 3`-

terminal C-residue; on single-stranded DNA;

2) phosphatase activity: Exo III removes the 3`-terminal

phosphate and generates a 3`-OH group;

3) RNase H activity: Exo III exonucleolytically degrades the

RNA strand in RNA/DNA hybrids;

4) Apurinic/apirimidinic-endonuclease activity: Exo III cleaves

phosphodiester bonds at apurinic or apyrimidinic sites to

produce 5`-termini that are base-free deoxyribose 5`-

phosphate residues.

Features:

1) the rate of DNA digestion by ExoIII depends upon temperature,

salt concentration and the molar ratio of DNA to enzyme in the

reaction mixture;

2) optimal reaction conditions should be determined experimentally.

Applications:

1) creation of unidirectional deletions in DNA fragments with

conjunction with S1 Nuclease;

2) generation of a single stranded template for dideoxy-sequencing of

DNA;

3) site-directed mutagenesis;

4) cloning of PCR ptoducts;

5) preparation of strand-specific probes.

10X Reaction buffer

660 mM Tris-HCl (pH 8.0 at 30

C), 6.6 mM MgCl

Inhibition and inactivation

Inhibitors: metal chelators, p-chloromercuri benzoate (50-90% inhibitory at

0.1 mM); inactivated by heating at 70

C for 10 min.

Fig. 9. Exonuclease III activity

DNA LIGASES

T4 DNA ligase

Catalyzes the formation of a phosphodiester between

juxtaposed 5`-phosphate and 3`-hydroxyl termini in duplex DNA or

RNA. The enxyme repairs single-strand nicks in duplex DNA, RNA

or DNA/RNA hybrids, joins DNA fragments with either cohesive or

blunt termini. The T4 DNA Ligase requires ATP as cofactor.

Features:

1) fast – sticky-end ligation is completed in 10 min at room

temperature;

2) active in buffers for restriction enzymes, PCR and RT (when

supplemented with ATP);

3) PEG solution provides efficient blunt-end ligation.

Applications:

1) cloning of restriction enzyme generated DNA fragments;

2) cloning of PCR products;

3) joining of double-stranded oligonucleotide linkers or adaptors to

DNA;

4) site-directed mutagenesis;

5) amplified fragment length polymorphism (AFLP);

6) ligase-mediated RNA detection;

7) nick repair in duplex DNA, RNA or DNA/RNA hybrids;

8) self-circularization of linear DNA.

10X T4 DNA ligase buffer

400 mM Tris-HCl), 100 mM MgCl

, 100 mM DTT, 5 mM ATP (pH 7.8 at

С).

Inhibition and inactivation

Inhibitors: is strongly inhibited by NaCl or KCl at>200 mM; inactivated by

heating at 65

С for 10 min or at 70

С for 5 min.

* Interesting details. Polyethylene glycol (PEG) greatly increases

the rate of ligation of blunt-end DNA; molecular weight – 55.3 kDa.

Taq DNA ligase

Taq DNA ligase catalyzes the formation of a phosphodiester bond

between juxtaposed 5´ phosphate and 3´ hydroxyl termini of two adjacent

oligonucleotides which are hybridized to a complementary target DNA. The

ligation will occur only if the oligonucleotides are perfectly paired to the

complementary target DNA and have no gaps between them; therefore, a

single-base substitution can be detected. TaqœDNA ligase is active at

elevated temperatures (45°C-65°C) .

Features:

1) Taq DNA ligase is not a substitute for T4 DNA ligase;

2) incorporates phosphorylated oligonucleotides duringœPCRœand

ligase chain reaction.

Applications:

1) allele-specific gene detection using ligase detection reaction and

ligase chain reaction;

2) mutagenesis by incorporation of a phosphorylated oligonucleotide

during primer extension amplification.

1X Taq DNA ligase buffer

20œmMœTris-HCl; 25œmMœpotassium acetate; 10œmMœmagnesium acetate;

1œmMœNAD; 10œmMœdithiothreitol; 0.1œ%œTriton X-100 (pH 7.6œat 25°C).

Inhibition and inactivation

Stability of Taq DNA ligase at 95°C:

0 min: 64,000 units/ml; 15 min: 32,000 units/ml; 60 min: 16,000 units/ml.

Fig. 10.

Taq DNA ligase

activity

Ligase

chain reaction

A DNA

amplification technique based upon the ligation of oligonucleotide

probes. The probes are designed to exactly match two adjacent

sequences of a specific target DNA. The chain reaction is repeated in

three steps in the presence of excess probe: (1) heat denaturation of

double-stranded DNA, (2) annealing of probes to target DNA, and

(3) joining of the probes by thermostable DNA ligase. After the

reaction is repeated for 20-30 cycles the production of ligated probe

is measured. o

Allele-specific LCR employs four oligonucleotides, two

adjacent oligonucleotides which uniquely hybridize to one strand of

target DNA and a complementary set of adjacent oligonucleotides,

hybridize to the opposite strand. Olugonucleotides from one cycle

serve as a matrix during next cycles.

Fig. 11. Scheme of ligase chain reaction

Technical data

Fig. I. DNA base pairs

Table I. Range of effective separation of DNA fragments in agarose gel

Agarose gel, % Length of DNA fragments, bp

0.5 2000-50000

0.6 1000-20000

1.0 400-8000

1.5 200-3000

3.0 25-1000

5.0 10-300

Table II. General physical constants

Name Constant

Avogadro`s number 6.022x10

mol

-1

Planck`s constant 6.626x10

-34

J·s

Boltzmann constant 1.38x10

-23

J·K

-1

Ideal gas constant 8.314 J/mol·K

Speed of light 2.9979x10

m/s

Atomic mass unit 1.66x10

-27

*Common conversions of oligonucleotides

Molecular weight:

MW =333xN

Concentration of oligonucleotides:

C (µM/ or pmol/µl) = A

260

/0.01xN;

C (ng/ml) = A

260

xMW/0.01xN;

MW – molecular weight, A

260

– absorbance at 260 nm, N – number of bases

*DNA/protein conversion

1 kb of DNA = 333 amino acid = 37 kDa

* Melting temperature of duplex DNA and oligonucleotides

For duplex oligonucleotides shorter than 25 bp, “The Wallace Rule”

(in

C) = 2(A+T) + 4(G+C), where

(A+T) – the sum of the A and T residues in the oligonucleotide and (G+C) –

the sum of G and C residues in the oligonucleotide.

For duplex DNA, <100 bp long

(in

C) = 81.5

C+16.6 (log

[Na

])+0.41(%[G+C]) –675/n –1.0m, where

n – thr number of bases in the oligonucleotide;

m – the percentage of base-pair mismathes

*Commonly used buffers

1 M Tris-HCl [tris(hydroxymethyl)aminomethane]:

Tris base 121.1 g

O to 800 ml

Adjust to desired pH with concentrated HCl.

Mix and add H

O to liter.

1X TBE (Tris-borate-EDTA) electropgoresis buffer:

Adjust to desired pH with concentrated HCl.

Tris base 108 g

Boric acid 55 g

0.5 M EDTA (pH 8.0) 40 ml

O to 1 liter

1X TE (Tris-EDTA) buffer, pH 7.4, 7.6 or 8.0, per liter:

1 M Tris, pH 7.4, 7.8, 8.0 108 g

0.5 M EDTA (pH 8.0) 2 ml

O to 1 liter

*Expressing concentrations of reagents

Molarity

M (mol/l) = number of moles solute/number of liters of solution

Percent composition

Concentration (%) = weight of solute (g)x100%/total weight of solution (g)

Ionic strength

The ionic strength, I, of a solution is a function of

the concentration of all ions present in that

solution.where c

is the molar concentration of ion

i (mol·dm

-3

), z

is the charge number of that ion,

and the sum is taken over all ions in the solution.

Table III. Genome sizes

Virus Length, nt Approx. MW, Da

Bacteriophage φX174 5,380 3.5x10

HIV 9,181 3.1x10

SARS Coronavirus 29,751 1x10

Bacteria

Agrobacterium tumefaciens

C58-Cereon

4.91x10

3.19x10

Staphylococcus aureus MW2 4.86x10

3.02x10

Streptococcus pneumoniae R6 2.04x10

1.33x10

Eukaryotes

Anopheles gambiae

PEST (malaria mosquito)

2.78x10

1.80x10

Caenorhabditis elegans

(round worm)

1.21x10

7.86x10

Drosophila melanogaster 1.37x10

8.9x10

Homo sapiens 3.15x10

2.07x10

Saccharomyces cerevisiae

S288C (budding yeast)

1.21x10

7.86x10

Plasmodium falciparum 3D7

(human malaria parasite)

2.29x10

1.49x10

Table IV. Atomic numbers and molecular masses of some chemical

elements

H C N O Na Mg P S Cl Ca

Atomic number 1 6 7 8 11 12 15 16 17 20

Atomic mass 1 12 14 16 23 24.3 31 32 35.4 40

Atomic mass – g/mol

Список использованной литературы (References)

1. Жимулёв И.Ф. Общая и молекулярная генетика. –

Новосибирск: Сиб. унив. изд-во, 2007. – 479 с.

2. Оберемок В.В. Методические рекомендации к

применению ПЦР-метода. – Симферополь: Таврический

национальный университет им. В.И. Вернадского, 2006. – 35 с.

3. Оберемок В.В. Генетики, молекулярные биологи и их

открытия. – Симферополь: Таврический национальный

университет им. В.И. Вернадского, 2008. – 35 с.

4. www. fermentas.com

5. www. invitrogen.com

6. www. synthesisgene.com

7. www. wikipedia.org

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науке.

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