Lecture+1+Molecular+cloning.pdf MOLECULAR BIOLOGY

molecualr cloning

1. Molecular Biotechnology II
Lecture 1
Molecular cloning

2. Molecular cloning
•To clone something is to make an identical copy, whether it is done by a
photocopy machine on a piece of paper, cloning Dolly the sheep, or
cloning DNA
•Cloning can also be considered an amplification process, in which we
currently have one copy and we want many identical copies
•Cloning DNA typically involves recombinant DNA. This also has a
simple definition: a DNA molecule from two (or more) different sources.

3. Recombinant DNA Cloning Procedure
1) Enzymatic
digestion

4. Recombinant DNA Cloning Procedure
2) Ligation of
Target and
vector
DNA Ligase

5. Recombinant DNA Cloning Procedure
3) Transform
Ligated DNA
into Bacteria

6. Major Steps in Gene Cloning
1. Isolation of the DNA of interest (insert DNA) that is to be put
in the vector.
2. A vector is obtained (usually commercially available).
3. The insert DNA and vector DNA are cut with restriction
enzymes that make compatible ends.
4. The insert DNA is joined by ligation (Ligase Enzyme) into the
vector.

7. Major Steps in Gene Cloning
5. The recombinant DNA construct (insert + vector) is transferred
into, and maintained in, a host cell (typically E. coli bacteria) by
the process of transformation.
6. The bacteria that have incorporated the foreign DNA are
identified and isolated from untransformed cells.
7. The cloned DNA can be manipulated so that the protein product
it encodes can be expressed by the host cell.
8. The vector replicates with the bacteria, producing identical copies
or clones of the insert DNA.

8. The insertion of a DNA fragment into a bacterial plasmid
with DNA ligase enzyme .
Major Steps in Gene Cloning

9. Purification and amplification of a specific DNA sequence by
DNA cloning in suitable host cells.
Major Steps in Gene Cloning

10. Tools of gene cloning
1.Restriction enzymes
2.Vector
3.T4 Ligase
4.Host cells

11. What are restriction enzymes?
1.Molecular scissors that cut double stranded DNA
molecules at specific points.
2.Found naturally in a wide variety of prokaryotes
3.An important tool for manipulating DNA.

12. Biological role
•Most bacteria use Restriction Enzymes as a defense mechanism
against bacteriophages.
•Restriction enzymes prevent the replication of the phage by cleaving
its DNA at specific sites.
•The host DNA is protected by Methylases which add methyl groups
to adenine or cytosine bases within the recognition site thereby
modifying the site and protecting the DNA.
What are restriction enzymes?

13. Restriction Enzymes
•Enzyme that cuts DNA at specific nucleotide sequences known
as restriction sites.
•Found in bacteria and have evolved to provide a defense
mechanism against invading viruses.
•In bacteria they selectively cut up foreign DNA in a process
called restriction
•To cut the DNA, restriction enzyme makes two incisions at each
strand of the DNA double helix.

15. Nomenclature of restriction enzymes
Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1

16. •Three different classes of restriction endonuclease are
recognized, each distinguished by a slightly different
mode of action.
•Types I and III are rather complex and have only a
limited role in genetic engineering.
•Type II restriction endonucleases, on the other hand,
are the cutting enzymes that are so important in gene
cloning.

17. Type II restriction endonucleases cut DNA at
specific nucleotide sequences
•Each enzyme has a specific recognition sequence at
which it cuts a DNA molecule.
•A particular enzyme cleaves DNA at the recognition
sequence and nowhere else.
•Recognition sites have symmetry (palindromic), A
palindromic sequence in DNA is one in which the 5’ to 3’
base pair sequence is identical on both strands.
•Each restriction enzyme cuts the recognition site in a
specific restriction site .
5’-GGATCC-3’
3’-CCTAGG-5’

18. •Restriction enzymes that have the same recognition
sequence as well as the same cleavage site are
Isoschizomers.
•Restriction enzymes that have the same recognition
sequence but cleave the DNA at a different site within
that sequence are Neochizomers. Eg:SmaI and XmaI
C C C G G G C C C G G G
G G G C C C G G G C C C
Xma I Sma I
Isoschizomers and Neochizomers

19. Blunt and sticky ends
•simple double-stranded cut in the middle of the recognition
sequence (Figure a), resulting in a blunt end or flush end
•With other enzymes the two DNA strands are not cut at
exactly the same position. Instead the cleavage is staggered,
usually by two or four nucleotides, so that the resulting
DNA fragments have short single-stranded overhangs at
each end (Figure 4.9b). These are called sticky or cohesive
ends together again

20. blunt end
sticky end

21. DNA Ligase
•DNA ligaseis a specific type of enzyme, that facilitates the
joining ofDNAstrands together by catalyzing the formation of
aphosphodiester bond.
•It plays a role in repairing single-strand breaks in
duplexDNAin living organisms, but some forms (such asDNA
ligase IV) may specifically repair double-strand breaks (i.e. a
break in both complementarystrands of DNA).
•In addition, DNA ligase has extensive use in molecular
biologylaboratories forrecombinant DNAexperiments
(gene cloning) to join DNA molecules together to
formrecombinant DNA.

23. Linkers and Adaptors
•Sticky ends are desirable for DNA cloning
experiments.
•Sticky ends are provided by treating the target
and vectors with the same R.E or with different
one producing the same sticky ends.
•But what if the target DNA is blunt ended
•So therefore we will have to use Linkers and
Adaptors.

24. Linkers
•Synthetic , Short and known double stranded
oligonucleotides sequence.
•Having blunted ends on both sides and R. Sites for
BamHI.
•Treatment with R.E produces sticky ends after ligation
with target DNA.

26. But what if target DNA also having the
same Restriction Site then?

27. Adaptors
•A Synthetic double stranded
Oligonucleotide having blunt end and
Sticky end.
•Blunt ends will bind to the blunt ends of
target DNA to produce new DNA with
sticky ends.
•Problems: sticky ends of adaptors will
bind with each other so….

28. •Adaptor molecules are synthesized so that the blunt end is the
same as “natural” DNA, but the sticky end is different. The 3′-OH
terminus of the sticky end is the same as usual, but the 5′-P
terminus is modified: it lacks the phosphate group, and is in fact
a 5′-OH terminus.
•DNA ligase is unable to form a phosphodiester bridge between
5′-OH and 3′-OH ends. The result is that, although base pairing is
always occurring between the sticky ends of adaptor molecules,
the association is never stabilized by ligation.
Adaptors

29. •Adaptors can therefore be ligated to a blunt-ended DNA
molecule but not to themselves. After the adaptors have
been attached, the abnormal 5′-OH terminus is
converted to the natural 5′-P form by treatment with the
enzyme polynucleotide kinase, producing a sticky-ended
fragment that can be inserted into an appropriate vector.
Adaptors

30. Homopolymer tailing (HT)
•Homopolymer: A strand composed of one type of
nucleotide.
•HT: the in-vitro addition of the same nucleotide by the
enzyme terminal deoxynucleotide transferase to 3’-OH
of a duplex DNA molecule. (calf thymus).
•e.g. Complimentary poly (C) and poly (G) for vector and
target DNA respectively.