Manipulation of purified DNA

♦ To produce recombinant DNA molecules, the vector and the DNA

to be cloned may have to be modified:

- shortened
- lengthened
- copied into new DNA molecules
- modified by the addition or removal of specific chemical groups

♦ Many of the DNA manipulation techniques make use of purified

enzymes, which within the cell participate in essential processes
such as DNA replication, transcription, repair, recombination,
breakdown of unwanted foreign DNA

♦ All the enzymes can be obtained from commercial suppliers

 Range of DNA manipulative enzymes

♦ DNA modifying enzymes:

- Nucleases (endonucleases, exonucleases)

- DNA polymerases
- Enzymes that act on termini

♦ Restriction endonucleases

♦ Ligases
Nucleases- degrade DNA molecules by breaking the
phosphodiester bonds that link one nucleotide to the next in a DNA
strand
♦ Exonucleases remove nucleotides one at a time from the end of a
DNA molecule
The enzyme Bal31 from Alteromonas espejiana removes nucleotides from both
strands of a double-stranded molecule
♦ Endonucleases break internal phosphodiester bonds within a
DNA molecule

S1 endonuclease from Aspergillus oryzae only cleaves single

strands
DNaseI or desoxyribonuclease I, which is prepared from cow
pancreas, cuts both single and double-stranded molecules. The end
result of prolonged DNaseI action is a mixture of very short
oligonucleotides
Polymerases are enzymes that synthesize a new strand of DNA
complementary to an existing DNA or RNA template

Most polymerases can function only if the template possesses a

double-stranded region that acts as a primer for initiation of
polymerization
Polymerases routinely used in Genetic Engineering:

- DNA polymerase I from E. coli

This enzyme attaches to a short single-stranded region (or nick) in a

mainly double-straded DNA molecule and synthesizes a completely
new strand, degrading the existing strand as it proceeds
DNA polymerase I has a dual activity- DNA polymerization and DNA
degradation

Nuclease aa 323 Polymerase

activity activity

X
Klenow fragment
Taq polymerase from Thermus aquaticus is used in the polymerase
chain reaction (PCR). This enzyme is thermostable, meaning that is
resistant to denaturation by heat treatment

Other polymerases proofreading (3´-5´exonuclease) commonly

used:

Pfu Pyrococcus furiosus

Pwo Pyrococcus woesi
Deep VentTM Pyrococcus sp.
Tma Thermotoga maritima
Reverse transcriptase is involved in the replication of several kinds
of virus. Uses RNA as a template. Is essential to the technique
called “complementary DNA (cDNA) cloning”
Enzymes that act on termini- they modify DNA molecules by
addition or removal of specific chemical groups

Alkaline phosphatase (from E. coli, calf intestinal tissue (CIP))

removes the phosphate group present at the 5´terminus of a DNA
molecule

Used to prevent recircularization of the vector DNA digested with a

specific restriction endonuclease
Terminal deoxynucleotidyl transferase from calf thymus tissue, adds
one or more deoxyribonucleotides onto the 3´terminus of a DNA
molecule

It is mainly used to add homopolymer tails to cDNA molecules in

order to clone them into a vector
The discovery and function of the restriction endonucleases

The initial observation was

made in the early 1950s,
when it was shown that some
bacteria are immune to
bacteriophage infection- host
controlled restriction
Understanding the biological basis of host-controlled restriction
and modification of bacteriophage DNA led to the identification
of restriction endonucleases

Host-controlled restriction
and modification of phage
l in E. coli strain K, by
efficiency of plating (EOP)
Enzymes for cutting DNA- Restriction endonucleases
♦Purified restriction endonucleases allow to cut DNA molecules in
the precise, reproducible manner required for gene cloning

♦Nobel prize for W. Arber, H. Smith and D. Nathans in 1978. It was

one of the key breakthroughs in the development of genetic
engineering

♦ They are synthesized by many, perhaps all species of bacteria

♦ Over 1200 different ones have now been characterized

♦ Four different types of restriction and modification (R-M) systems
have been recognized but only one (Type II) is widely used in gene
manipulation

The naming of restriction endonucleases provides

information about their source
Type II restriction endonucleases cut DNA at specific
nucleotide sequences

♦ A particular enzyme cleaves DNA at its recognition sequence and

nowhere else

PvuI and PvuII from Proteus vulgaris cut at the hexanucleotides

CGATCG and CAGCTG, respectively

♦ Many restriction enzymes recognize hexanucleotide target sites,

but other cut at four, five eight or longer nucleotide sequences. They
all have a twofold axis of symmetry- palindromes

♦ There are also restriction endonucleases with degenerate

recognition sequences. Ex: HinfI recognizes GANTC (GAATC;
GATTC; GAGTC; GACTC)
♦ Restriction enzymes with the same specificity are known as
isoschizomers
Blunt ends and sticky ends

The exact nature of the cut produced by a restriction endonuclease

is important in the design of the cloning experiment

♦ Some restriction enzymes make a double-stranded cut in the

middle of the recognition sequence, resulting in a blunt end
♦ Many restriction endonucleases cut both strands at a
distance of 2 or 4 nucleotides and the resulting DNA fragments
have short single-stranded overhangs at each end- sticky or
cohesive ends
♦ Cohesive ends of DNA fragments produced by digestion with
EcoRI
Restriction endonucleases with different recognition sequences
may produce the same sticky ends
Performing a restriction digest in the laboratory

Buffer for restriction of

DNA:

Tris pH7.4, MgCl2, NaCl,

Dithiothreitol
Separation of DNA molecules by
gel electrophoresis

Estimation of the sizes of

DNA molecules
Mapping the positions of different restriction sites in
a DNA molecule

For cloning purposes is

important to have a
restriction map so that
the correct restriction
endonucleases can be
selected
 How to construct a restriction map?
- A series of restriction digests must be performed

- The number and sizes of the fragments produced by each

endonuclease must be determined

- Series of double and triple digestions

- Assembly of the fragments in the correct order

 Ligation- joining DNA molecules together

Ligation is the final step in construction of a recombinant DNA

molecule
 The mode of action of the ligase

♦ Within the cell the enzyme carries out the repair of

discontinuities that may arise in one of the strands of a
double-stranded molecule. This lack of phosphodiester bond
may arise by breakage of the cell´s DNA molecules or during
DNA replication and recombination

♦ Ligase used in Genetic Engineering is purified from E. coli

infected with the phage T4

♦ In the test tube, purified DNA ligases can join together

individual DNA molecules or the two ends of the same
molecule
♦ Blunt end ligation is not very efficient because ligase has to
wait for chance associations to bring the ends together. Blunt
end ligation should be performed at high DNA concentrations

♦ Ligation is performed at 16 ºC
♦ Ligation of complementary sticky ends is much more
efficient, because the base-paring between the two molecules
is a relatively stable structure for the enzyme to work on
Putting sticky ends onto a blunt-ended molecule

Producing sticky
ends by
homopolymer
tailing
(Cont.)