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simplicity of PCR allowed this method to be used in different

branches of genetic investigations. Natural process of cell replication

lies in the base of PCR.

Every cycle consists of three steps: denaturation, annealing of

primers, synthesis of DNA strand. For every step there is special

temperature and time interval:

1) denaturation (94

C, 0.5-1 min);

2) annealing of primers (35-65

C, 0.5-1 min);

3) synthesis (72

C, 0.5-1 min).

Accurate extraction of DNA, amplification of DNA, and detection

of amplification products (electropheresis) provide high quality polymerase

chain reaciton. Amount of amplicons of DNA fragment from one cell

created during PCR can be foumd with following formula: F = 2

, n-number

of cycles.

Fig. 3. Scheme of PCR: I, II, III, – 1

, 2

and 3

cycles; 0 –

fragment of target DNA; a – denaturation; b – annealing of primers; c –

synthesis

DNA sequencing

Is the method for determining the order of the nucleotide

bases in target DNA. As a matrix in DNA sequencing single strand

DNA is used. To initiate DNA sequencing with DNA polymerase

synthetic primers or natural subfragments got with restriction

endonucleases are used. 3’-end of primer must end before DNA part

that wil be sequenced. This method was invented by UK scientist

Frideric Sanger in 1975 (dideoxy termination method).

Fig. 4. Scheme of DNA sequencing

To reveal sequence of nucleotides in target part of DNA, perform 4

polymerase reations. In each of four reaction mixtures is obligatory to add

deoxynucleotide triphosphates (dCTP, dGTP, dTTP, dATP) and DNA

polymerase. Beside this, in each of tubes one of dideoxynucleotide

triphosphates (ddCTP, ddGTP, ddTTP и ddATP) is added. ddNTP in 3’-

position instead of hydroxyl group (OH) contains hydrogen (Н). ddNTP

takes part in DNA polymerisation reaction and ends it. In each reaction

mixture proportion between dCTP, dGTP, dTTP, dATP and one of ddNTP

is 100:1. Because of low concentration of ddNTP polymerase reaction does

not end in the beginning of DNA sequencing. At the same time reaction

mixture contains big amount of DNA fragments of different length (because

of ddNTP connection at different points of growing strand).

Using electrophoresis products of DNA polymerisation are

separated and get sequence of target DNA.

ENDONUCLEASES

DNase I

Is an endonuclease that digests single- and double-stranded

DNA. It hydrolyzes phosphodiester bonds producing mono- and

oligodeoxyribonucleotides with 5`-phosphate and 3`-OH groups.

Features:

1) the enzyme activity is strictly dependent on Ca

and is

activated by Mg

or Mn

ions;

2) in the presence of Mg

, DNase I cleaves each strand of

dsDNA independently, in a statistically random fashion;

3) in the presence of Mn

ions, the enzyme cleaves both DNA

strands at approximately the same site, producing DNA

fragments with blunt-ends or with overhang termini of only

one or two nucleotides;

4) DNase is sensitive to physical denaturation, that is why mix

gently by inverting the tube, do not vortex.

Applications:

1) preparation of DNA-free RNA;

2) removal of template DNA following in vitro transcription;

3) preparation of DNA-free RNA prior to RT-PCR;

4) DNA labeling by nick translation in cojunction with DNA

Polymerase;

5) studies of DNA-protein interactions by DNase I;

6) generation of a library of randomly overlapping DNA inserts.

Reaction buffer containing Mn

is used.

10X Reaction buffer with MgCl

100 mM Tris-HCl (pH 7.5 at 25

C), 25 mM MgCl

1 mM CaCl

10X Reaction buffer with MnCl

100 mM Tris-HCl (pH 7.5 at 25

C, 1 mM CaCl

Recommended

concentration of MnCl

in 1X reaction buffer is 10 mM.

Inhibition and inactivation

Inhibitors: metal chelators, transition metals, (e.g., Zn) in milimolar

concentrations, SDS (even in concentrations less than 0.1 %), reducing

agents (DTT), ionic strength above 50-100 mM; inactivated by by heating

at 65

C for 10 min in the presence of EDTA (use at least 1 mol of EDTA

per 1 mol of Mg

/Mn

Fig. 5. DNase I activity in the presence of Mg

or Mn

Endonuclease IV, E. coli (Endo 4)

Endonuclease IV recognizes apurinic/apirimidinic (AP) sites

of dsDNA and cleaves phosphodiester bond 5` to the lesion

generating a hydroxyl group at the 3`-terminus. The enzyme can also

act as esterase releasing a 3`-phosphoglycolate or 3`-phosphate from

the damaged ends of dsDNA. Endo IV possesses also a 3`→5`

exonuclease activity.

Features:

1) its progression on substrates is sensitive to ionic strength, metal

ions, EDTA, and reducing conditions;

2) substrates with 3`-recessed ends are preferred substrates for the

3`→5` exonuclease activity;

3) the enzyme has no requirement for Mg

but is more active in the

presence of Mg

Applications:

1) studies of DNA damage and repair;

2) single cell electrophoresis;

3) antitumour drug research;

4) DNA structure research;

5) SNP analysis.

10X Reaction buffer

100 mM Tris-HCl (pH 8.5 at 25

C), 25 mM MgCl

Inhibition and inactivation

Inhibitors: the enzyme is fairly resistant to EDTA during the reaction, but

becomes sensitive to even sub milimolar quantities of chelators when no

DNA substrate is present; inactivated by heating at 80

C for 15 min.

Fig. 6. Endonuclease IV activity

Restriction enzymes

Restriction enzymes recognize specific nucleotide sequences

and cleave DNA molecules either within or outside their recognition

site. Digestion results in the degeneration of DNA with “sticky” (5`-

or 3`-overhang or “blunt” ends).

Common properties

The phenomenon of host specificity was first observed by Luria

and Human in the early 1950s. Nearly a decade later, Arber and and Dussoix

predicted its molecular basis. They proposed that host specificity in two-

enzyme system: a restriction enzyme, which recognizes specific DNA

sequences and cleaves foreign DNA upon its entrance into the bacterial cell,

and a modification enzyme (metyltransferase), which protects the host DNA

from degradation by its own restriction enzyme. Both restriction and

modification enzymes recognize the same nucleotide sequence and together

they form a restriction-modification (R-M) system.

Restriction-modification systems are wide-spread among bacteria

and have also been isolated from phage, Archaea and eukaryotic algae

viruses. R-M systems have been classified into four types (I, II, III and IV)

depending on the complexity of their structure, cofactor requirements, mode

of action and sequence cleaved. The best characterized and the most

frequently used are type II restriction endonucleases. These enzymes

recognize specific 4-8 bp DNA sequences and cleave a DNA sequence

either within the recognition site, or at a specified position up to 20 base

pairs outside the site.

Often there is more than one enzyme that recognizes a particular

nucleotide sequence. According to restriction enzyme nomenclature, an

enzyme with a unique specificity which has been discovered first is called

prototype. Subsequently discovered enzymes with the same specificity are

termed isoschisomers. An isoschisomer may differ from the prototype in site

preferences, reaction conditions, as well as in sensitivity to methylation and

exhibition of star activity. Restriction enzymes that recognize the same

nucleotide sequence, but cleave DNA at different positions are called

neoschisomers.

Site preferemces by restriction endonucleases

In 1975, Thomas and Davis discovered that EcoRI cleaves its five

recognition sites on phage λ DNA at rates that differ by an order of

magnitude. Similar examples have been documented for other restriction

enzymes. Factors such as flanking sequences, methylation of DNA and the

number of cleavage sites appear to influence cleavage efficiency. Cleavage

of resistant sites can be significantly enhanced by the addition of cleavable

DNA and oligodeoxyribonucleotide containing the recognition site, or

spermidine.

Types of restriction enzymes:

1) type I enzymes cleave at sites remote from recognition site; require

both ATP and S-adenosyl-L-methionine to function;

multifunctional protein with both restriction and methylase

activities;

2) type II enzymes cleave within or at short specific distances from

recognition site; most require magnesium; single function

(restriction) enzymes independent of methylase;

3) type III enzymes cleave at sites a short distance from recognition

site; require ATP (but doesn't hydrolyse it); S-adenosyl-L-

methionine stimulates reaction but is not required; exist as part of a

complex with a modification methylase;

4) type IV enzymes target methylated DNA.

Star activity

Restriction enzymes recognize specific nucleotide sequences within

DNA molecules. However their recognitionsite specificity can be reduced in

vitro. Under certain conditions, enzymes are able to recognize and cleave

nucleotide sequences which differ from the canonical site. At low ionic

strength, for example, BamHI (with the recognitionsequence GGATCC) is

able to cleave the following sequences: NGATCC, GPuATCC and

GGNTCC. This phenomenon is called “relaxed” or “star” activity. For most

restriction enzyme applications, star activity is not desirable.

Star activity is the result of:

1) prolonged incubation (over digestion);

2) high enzyme concentration in the reaction mixture;

3) high glycerol concentration in the reaction mixture;

4) presence of organic solvents, such as ethanol, dimethyl sulfoxide

(DMSO) or dimethyl formamide (DMFA), in the reaction mixture;

5) low ionic strength of the reaction buffer;

6) suboptimal pH values of the reaction buffer;

7) substitution of Mg

for other divalent cations, such as Mn

In some cases, the termini generated by DNA cleavage with a

restriction enzyme at the canonical site have been shown to stimulate

enzyme star activity.

Both star activity and incomplete DNA digestion result in atypical

electrophoresis patterns that can be distinguished by careful examination of

gel images.

Differences between star activity and incomplete digestion

Star activity results in additional DNA bands below the expected

bands below the expected bands and no additional bands above the largest

expected fragment. These additional bands become more intense, while the

expected bands become less intense, when either the incubation time or the

amount of enzyme is increased.

Incomplete DNA digestion results in additional bands above the

expected DNA bands on the gel. Additional bands disappear when the

incubation time or amount of enzyme is increased. No additional bands

below the smallest expected fragment are observed.

Certain restriction enzymes (for example, Alol, Taul, EcoRII)

remain associated with the substrate DNA after cleavage and cause a change

in the mobility of the digestion products during electrophoresis. In these

cases SDS is added, solution is heated for 10 min at 65

C and chill on ice

prior to loading on the gel.

Digestion of methylated DNA

DNA methylation is the process of transferring a methyl group

from a donor molecule to either cytosine or an adenine by DNA

methyltransferases. Such methylation is the most common and abundant

DNA modification process in living organisms. Thre types of methylated

bases are predominately found in DNA: 1) 5`-methylcytosine (m5C); 2) N4-

methylcytosine (m4C); 3) N6-methyladenine (m6A).

In prokaryotes, DNA cleavage by a cognate restriction enzyme is

prevented by the methylation of DNA by a sequnce-specific

methyltransferase which is an integral component of every restriction-

modification (R-M) system. The majority of E. coli strains used for

propagation of plasmid DNA contain two-site specific DNA

methyltranferases – Dam and Dcm. The methylase encjded by the dam gene

methylates the N6-position of an adenine residue with the GATC sequence.

The methylase encoded by the dcm gene methylates the C5-position of the

internal cytosine residue within the CCWGG sequence.

DNA from higher eukaryotic organisms possesses modified 5-

methylcytosine residues within CpG or CpNpG contexts. These tissue-

specific methylation patterns are heritable. They participate in regulation of

gene expression and cellular differentiation.

In cases where a restriction enzyme target site overlaps a

methylation site, the following digestion results are possible: 1) no effect; 2)

partial inhibition; 3) complete block.

To achieve effective DNA digestion, it is necessary to take into

account both the type of DNA methylation and the sensitivity of the

restriction enzyme to the type of methylation.

Sigle letter code (for describing sites of restriction enzymes)

R = G or A; Y = C or T; W = A or T; M = A or C; K = G or T; S = C or G;

H = A, C or T; V = A, C or G; B = C,G, or T; D = A, G or T; N = G, A, T or

C (A, T, G, C – nitrogen bases).

Examples of restriction enzymes

Alul

Dam, Dcm, CpG – no effect. Becomes inactivated

if heated 20 min at 65

C. 4 bp from end of DNA is

required for complete digestion. The optimal

incubation temperature is 37

*Interesting details. Generally restriction enzymes are active in

PCR buffer.

XceI (NspI)

Dam, Dcm, CpG – no effect. Becomes inactivated

if heated 20 min at 65

C. The optimal incubation

temperature is 37

Tail (MaeII)

To ensure efficiency of digestion, perform the

cleavage reaction under paraffin oil. Optimal

temperature – 65

C. If methylation occurs in CpG

(in cytosine) site the cleavage is blocked ("CpG" is

shorthand for "-C-phosphate-G-", that is, cytosine and guanine separated by

a phosphate, which links the two nucleosides together in DNA). Can not be

inactivated if heated . 2 bp from end of DNA is required for complete

digestion. The optimal incubation temperature is 65

*Interesting details. H.O. Smith, K.W. Wilcox, and T.J. Kelley,

working at Johns Hopkins University in 1968, isolated and characterized the

first restriction nuclease whose functioning depended on a specific DNA

nucleotide sequence. Working with Haemophilus influenzae bacteria, this

group isolated an enzyme, called HindII, that always cut DNA molecules at

a particular point within a specific sequence of six base pairs. This sequence

is: 5' G T (T or C) ( A or G) A C 3'

3' C A (A or G) (T or C) T G 5'

They found that the HindII enzyme always cuts directly in the

center of this sequence. Wherever this particular sequence of six base pairs

occurs unmodified in a DNA molecule, HindII will cleave both DNA

strands between the 3rd and 4th base pairs of the sequence.

Restriction

analysis

This method is

based on the ability of

restriction enzymes to

find and cleave

specific sites of DNA.

Fig. 7. Scheme of restriction analysis

If change in site sequence occurs (mutatation, recombination,

methylation of DNA, etc.) a restriction enzyme loses its ability to

carry its function. The basic idea is: different sequences of DNA lead

to cutting DNA with restriction enzymes in different sites. Got with

restriction enzymes DNA fragments serve as markers of different

processes that take place inside the cells of investigated organism.

*Lab Shelf

PEG – polyethylene glycol. PEG is used in a

number of toothpastes as a dispersant. PEG is a

commonly used as precipitant for plasmid DNA

isolation and protein

crystallization. When

attached to various

protein medications,

polyethylene glycol

allows a slowed clearance of the carried protein

from the blood. It replaces water in wooden

objects, which makes the wood dimensionally

stable and prevents warping or shrinking of the

wood when it dries. In the field of microbiology,

PEG precipitation is used to concentrate

viruses.

Water - no tea in the evening without it.