Recombinant DNA Technology

3. overview
Recombinant DNA technology
Restriction endonucleases
Vectors
DNA cloning
Polymerase chain reaction
Blotting techniques

4. Recombinant DNA technology
1. Recombinant DNA technology is genetic
engineering which effects artificial
modification of the genetic constitution of a
living cell by introduction of foreign DNA
through experimental techniques.

5. Tools of Recombinant DNA
technology
.
Restriction endonucleases
Cloning of DNA
Probes

6. ENZYMES
 Restriction endonucleases
 Exonucleases
 Endonucleases
 Reverse transcriptase
 DNA polymerases
 DNA ligase

10. palindrome

11. Restriction endonucleases
Special group of bacterial enzymes which cleave
double stranded DNA into smaller more manageable
fragments.
Restriction endonucleases cut the DNA at the
palindome , which is short stretch of DNA (4-6 bp)
which exhibit two fold symmetry.

12. A restriction enzyme is named according to the
organism from which it was isolated. Hae111
Sticky and blunt ends
Restriction site is the DNA sequence recognized by a
resrictriction enzyme.

13. vectors
It’s a molecule of DNA to which the fragment of
DNA to be cloned is joined.
It’s a molecule of DNA to which the fragment of
DNA to be cloned is joined.

14. Types of vectors
1. Bacterial plasmids
2. Bacteriophage lambda
3. Cosmids
4. Retroviruses

17. Essential properties of a vector:
It must be capable of autonomous replication within
a host cell.
 It must contain at least one specific nucleotide
sequence recognized by a restriction endonuclease.
it must carry at least one gene that confers the ability
to select for the vector, such as an antibiotic
resistance gene.

18. plasmids:
Prokaryotic organisms contain single, large,
circular chromosome.
 In addition, most species of bacteria also
normally contain small, circular, extra-
chromosomal DNA molecules called plasmids.
 Plasmid DNA undergoes replication that may or
may not be synchronized to chromosomal
division.

19. Plasmids may carry genes that convey antibiotic
resistance to the host bacterium, and may
facilitate the transfer of genetic information
from one bacterium to another.
The plasmids are the most commonly used
vectors and can accept short DNA pieces about 6
to 10 kb long.

23. DNA CLONING
A clone is a large population of identical
molecules, bacteria or cells that arise from a
common ancestor.
Introduction of a foreign DNA molecule into a
replicating cell permits the amplification (that is,
production of many copies) of the DNA.

25. Steps of cloning
To clone a nucleotide sequence of interest, the
total cellular DNA is first cleaved with a specific
restriction enzyme, creating hundreds of
thousands of fragments.
Each of the resulting DNA fragment is joined to a
DNA vector molecule (known as a cloning vector)
to form a hybrid molecule

26. Each hybrid recombinant DNA molecule conveys
its inserted DNA fragment into a single host cell,
for example, a bacterium, where it is replicated
(or "amplified").
As the host cell multiplies, it forms a clone in
which every bacterium carries copies of the same
inserted DNA fragment, hence, the name
"cloning."

27. The cloned DNA is eventually released from its
vector by cleavage (using the restriction
endonuclease) and is isolated. By this
mechanism, many identical copies of the DNA of
interest can be produced.

28. recap
Recombinant DNA technology
Restriction endonucleases
Vectors (plasmids)
Probes
DNA cloning

29. probes
A single stranded piece of DNA labelled with a
radioisotope or antibiotic such as biotin.
The nucleotide sequence of the probe is
complementary to the gene of interest, called the
target DNA.
Probes therefore identify which band on gel
contains the target DNA.

30. Polymerase chain reaction
The polymerase chain reaction (PCR) is an
enzymatic (test tube, in vitro) method for
amplifying a selected DNA sequence that does
not rely on the biologic cloning method.
PCR permits the synthesis of millions of copies
of a specific nucleotide sequence in a few hours.

32. PCR uses DNA polymerase to repetitively amplify
targeted portions of DNA.
Each cycle of amplification doubles the amount
of DNA in the sample.
leads to an exponential increase in DNA with
repeated cycles of amplification.

33. Steps of PCR
1. Primer construction
It is not necessary to know the nucleotide
sequence of the target DNA in the PCR method.
However, it is necessary to know the nucleotide
sequence of short segments on each side of the
target DNA.

34. These stretches, called flanking sequences,
bracket the DNA sequence of interest.
The nucleotide sequences of the flanking regions
are used to construct two, single stranded‑
oligonucleotides, usually 20 to 35 nucleotides
long, which are complementary to the respective
flanking sequences. These serve as primers.

35. 2. Denature the DNA:
The DNA to be amplified is heated to separate
the double stranded target DNA into single‑
strands.

36. 3. Annealing of primers to
single stranded DNA:‑
 The separated strands are cooled and allowed to
anneal to the two primers (one for each strand).

37. 4. Chain extension:
DNA polymerase and deoxyribonucleoside
triphosphates (in excess) are added to the
mixture to initiate the synthesis of two new
chains complementary to the original DNA
chains.
 DNA polymerase adds nucleotides to the
3' hydroxyl end of the primer, and strand growth‑
extends across the target DNA, making
complementary copies of the target.

38. At the completion of one cycle of replication, the
reaction mixture is heated again to denature the
DNA strands (of which there are now four).
Each DNA strand binds a complementary primer,
and the cycle of chain extension is repeated.

39. By using a heat stable DNA polymerase from a‑
bacterium that normally lives at high
temperatures (thermophilus aquaticum), the
polymerase is not denatured and, therefore, does
not have to be added at each successive cycle.
Typically twenty to thirty cycles are run during
this process, amplifying the DNA by a
million fold to a billion fold.‑ ‑

40. Advantages of PCR:
The major advantages of PCR over cloning as a
mechanism for amplifying a specific DNA
sequence are sensitivity and speed.
DNA sequences present in only trace amounts
can be amplified to become the predominant
sequence

41. APPLICATIONS OF PCR
Comparison of a normal gene with an mutant
form of the gene for detection of mutations.
PCR allows the synthesis of mutant DNA in
sufficient quantities for a sequencing protocol
without laboriously cloning the altered DNA.

42. 2. Detection of low—abundance
nucleic acid sequences
For example, viruses that have a long latency
period, such as HIV, are difficult to detect at the
early stage of infection using conventional
methods.
 PCR offers a rapid and sensitive method for
detecting viral DNA sequences even when only a
small proportion of cell’s is harboring (shelter)
the virus.

43. 3. Forensic analysis of DNA
samples:
DNA fingerprinting by means of PCR has
revolutionized the analysis of evidence from
crime scenes.
 DNA isolated from a single human hair, a tiny
spot of blood, or a sample of semen is sufficient
to determine whether the sample comes from a
specific individual.

44. 4 verification of paternity
Utilizes the same technique of DNA fingerprinting .

45. 4. Prenatal diagnosis and carrier
detection cystic fibrosis:
Cystic fibrosis is an autosomal recessive genetic
disease resulting from mutations in the cystic
fibrosis trans-membrane regulator (CFTR) gene.
Because the mutant allele is three bases shorter
than the normal allele, it is possible to
distinguish them from each other by the size of
the PCR products obtained by amplifying that
portion of the DNA.

46. Uses of recombinant DNA
technology
Diagnostic purpose as in PCR.
Treatment purpose
 vaccines
 hormones
 factor 8 , TPA (tissue plasminogen activator).
Genetic counselling
Gene therapy (SCID).
Transgenic animals

47. Southern blotting:
Southern blotting is a technique that can detect
mutations in DNA.
Experimental procedure: This method, named
after its inventor, Edward Southern, involves the
following steps.

49. Steps
First, DNA is extracted from cells, e.g., a patient's
leukocytes.
 The DNA is cleaved into many fragments using a
restriction enzyme.
The resulting DNA fragments are separated on
the basis of size by electrophoresis.

50. The DNA fragments in the gel are denatured into
single strands and transferred to a nitrocellulose
membrane for analysis.
A radioisotope labelled probe is used to identify
the gene of interest.

52. Northern blots:
Northern blots are very similar to Southern
blots, except that the original sample contains a
mixture of mRNA molecules that are separated
by electrophoresis, then transferred to a
membrane and hybridized to a radioactive probe.
The bands obtained by autoradiography give a
measure of the amount and size of particular
mRNA molecules in the sample.

54. Western blots:
Western blots (also called immunoblots) are
similar to Southern blots, except that protein
molecules in the sample are separated by
electrophoresis and blotted to a membrane. The
probe is a labeled antibody, which produces a
band at the location of its antigen.