Importance of gene cloning: obtain pure Slide2

DNA of interest for study

Fragment to be cloned /Gene of interest (G.o.i)
belongs to a complex mixture of DNA fragments –
entire complement of a genome.

---Each of fragment is inserted into a vector-

multiple rDNAs

---Usually one rDNA is trnsported into one host

cell - were each is amplified many copies –
rDNA clones- plate them out on media -
colonies
--Each colony has a multiple bacteria in which
each bactera has one type of rDNA in more than
one copy

--- So from a complex mixture of fragments now

ur fragment/gene of interest is amplified in a live
bacteria.- What is problem now?

----multiple bacterial clones> which is ur clone of

interest?
Importance of gene cloning: obtain pure
DNA of interest for study
Problem of selection
Slide3
Genome from which one gene is to be
studied .
E.Coli has approximately 4000 genes
---fragments cloned are so many….so many
rDNA > so many bacterial clones.> one
clone
--use different sstragtegies to ensure u have
right bacterial clone----problem of selection
-1) cloning strategy is modified so cells with
desired rDNA can grow
2) Techniques help identify one clone from
many.
Importance: one colony> DNA of interest is
cloned/ amplified> isolate pure DNA from it
for express and study structure and
function.
PCR Slide 4
---Here reaction tube having DNA is mixed
with a set of reagents and incubated in a PCR
instrument called thermocycler
enables the mixture to be incubated at a series
of temperatures that are varied in a
preprogrammed manner.
1. 94C: denature the ds DNA by breaking H
bonds
2. 50-60: cool down the mixture which have
excess of short oligonucleotides or primers.
They will bind to specific regions on DNA.
3. 74C: Taq polymerase will attach to one end
of the primer and synthesize DNA.
4. 94C: denature-anneal-elongate

---In two cycles see 2^2 = 4 strands ,

30x-130 million new ds DNA . Amplified the
original dsDNA.
PCR Slide 5

Importance of PCR: by designing primers specific to delinating positions on the

template of interest e.g. If primer are annealing to either side of the gene > many
copies of the gene will be made.

Outcome of PCR is same as gene cloning. You again made copies of the DNA of
interest.

Benefit :
1.Just ur desired DNA is amplified and automatically selected and so unlike in gene
cloning there is no problem of selection.
2. Completed in few hours, gene cloning takes days

Limitations of PCR:
1. Sequences of the annealing site of the primer must be known. It cannot be used
to isolate genes which have not been studied before whose sequences are not
known.
2. is a limit to the length of DNA sequence that can be copied by PCR. Upto 5 kb,
with special techniques upto 40kb.

HOWEVER, in veretebrates, genes can be longer than 40 knb and so gene cloning is
a way to isolate long genes or those not studied before.
Applications Slide 6

---Isolate or detect known sequences-diagnostics

--PCR of human globin genes, for example, is used to test for the presence of
mutations that might cause the blood disease called Thalassaemia
--After the PCR, the gene copies are sequenced or
studied in some other way to determine if any of the thalassaemia mutations are
present.

---DNA of a disease-causing virus- positive result indicates that a sample contains the
virus and person is infected and needs treatment…..>
----tremendously sensitive>
even if there is just one DNA molecule in the starting mixture it can detect
--early detection of virus infectio
--forensics-material such as
hairs and dried bloodstains or even from the bones of long-dead humans
DNA manipulative enzymes Slide7
Pure samples of DNA have been prepared:
Manipulate it with pure enzymes by methods such as: cut, join, shorten,
lengthen DNA, copy it into RNA or new molecules of DNA, add ssome chemical
groups.
Cutting –restriction endonucleases
Join- ligases.
DNA manipulative enzymes: four broad classes:
1. Nucleases are enzymes that cut, shorten, or degrade nucleic acid molecules.
2. Ligases join nucleic acid molecules together.
3.Polymerases make copies of molecules.
4.Modifying enzymes remove or add chemical groups.
few display multiple activities that span two or more classes: many
polymerases combine their ability to make new DNA molecules with an
associated DNA degradative (i.e., nuclease) activity
Nucleases degrade DNA molecules by breaking the phosphodiester bonds that link
one nucleotide to the next in a DNA strand. There are two different kinds of Slide 8
nuclease:
l Exonucleases remove nucleotides one at a time from the end of a DNA molecule.
l Endonucleases are able to break internal phosphodiester bonds within a DNA
molecule.
Classification of exonucleases: Distinction between different exonucleases
---the number of strands that are degraded when a double-stranded molecule is
attacked exonuclease
• that removes nucleotides from both strands of a double-stranded molecule
-- Bal31 purified from the bacterium Alteromonas espejiana (reaction: greater the
---------over the length of time that Bal31 will yeild shorter ds DNA fragments
• Enzymes such as E. coli exonuclease III degrade just one strand of a double-
stranded molecule from the 3’end, leaving single-stranded DNA as the product
Classification of endonucleases:
• cleaves single strands-S1 endonuclease (from the fungus Aspergillus oryzae)
• cuts both single and double-stranded molecules DNase I, which is prepared from
cow pancreas. DNase I is non-specific in that it attacks(reaction mixture -action is
a mixture of mononucleotides and very short oligonucleotides.)
DNA at any internal phosphodiester bond, restriction endonucleases: cleave
doublestranded DNA only at a limited number of specific recognition sites
Exonuclease Slide 9
from Bal31from
Alteromonas espejiana

Both ends/strands of dsDNA

both at 5’ and 3’ end

E. coli exonuclease III –

Endonuclease 3’ to 5’ direction
Endonuclease Slide 10

S1 nuclease, fungus Aspergillus oryzae)

which cleaves only single-stranded
DNA, including
single-stranded nicks in mainly
double-stranded molecules.
Leaves behind 5’ mononucleotides

b) DNase I, (cow pancreas )

which cleaves both single- and
double-stranded DNA.
---mixture of mononucleotides and v
ery short oligonucleotides

c) A restriction endonuclease, which

cleaves double-stranded DNA, but
only at a limited number of sites.
Ligases Slide 11

LIGASES : Cellular function of DNA ligase is to repair

single-stranded breaks (“discontinuities”) that arise
in double-stranded DNA molecules during,
for example, DNA replication.

DNA ligases from most organisms can also join together

two individual fragments of double-stranded DNA
 Slide 12
POLYMERASES: DNA polymerases are enzymes that synthesize a new strand of DNA
complementary to an existing DNA or RNA template in 5’ >3’
----the template – a single stranded
---- primer possesses a double-stranded region required for initiation of
polymerization.
Four types of DNA polymerase are used routinely in genetic engineering:
1. DNA polymerase I, which is usually prepared from E. coli. This enzyme attaches to a short
single-stranded region (or nick) in a mainly double-stranded DNA molecule, and then
synthesizes a completely new strand, degrading the existing strand as it proceeds. DNA
polymerase I is therefore an example of an enzyme with a dual activity—DNA
polymerization and DNA degradation.
2. Klenow fragment: polymerase and nuclease activities of DNA polymerase I: different parts
of the enzyme molecule. The nuclease activity is contained in the first 323 amino acids of the
polypeptide, so removal of this segment leaves a modified enzyme that retains the
polymerase function but is unable to degrade DNA. This modified enzyme Is the klenow
fragment. synthesize a complementary DNA strand on a single-stranded template, but as it
has no nuclease activity it cannot continue the synthesis once the nick is filled in .
Application of these polymerases is in DNA sequencing.
Taq DNA polymerase: Thermus aquaticus. This organism lives in hot springs, and many of its
enzymes, including the Taq DNA polymerase, are thermostable, meaning that they are
resistant to denaturation by heat treatment.--94°C to denature the DNA.
3. Reverse transcriptase, an enzyme involved in the replication of several kinds of virus.
template not DNA but RNA--complementary DNA (cDNA) cloning.
 Slide 13

The basic reaction: a new DNA strand is synthesized in the 5′ to 3′ direction.

(b) DNA polymerase I, which initially fills in nicks but then continues to synthesize a new strand,
degrading the existing one as it proceeds.

(c) The Klenow fragment, which only fills in nicks.

(d) Reverse transcriptase, which uses a template of RNA.

DNA modifying enzymes (a) Alkaline phosphatase, (from E. coli, calf Slide 14
intestinal tissue, or arctic shrimp),, which removes
5′-phosphate groups.

(b) Polynucleotide kinase, (from E. coli infected with T4 phage),

which attaches 5′-phosphate groups

single-stranded or

double-stranded molecules.

Terminal deoxynucleotidyl transferase, (from calf thymus tissue), which attaches

deoxyribonucleotides to the 3′ termini of polynucleotides in either
Restriction endonucleases: Gene cloning requires very precise and reproducible cuts in DNA.
Slide 15
construction of a recombinant DNA molecule
molecule-cleaved at a single position, to open
up the circle> new DNA can be inserted

Vectors also have a size limit for

carrying the new DNA molecule.

1978: Nobel Prizes for W. Arber, H. Smith, and D. Nathans:-discovered restriction endonucleases
which allow DNA molecules to be cut in the precise, reproducible manner required for gene cloning.
Discovery of restriction endonucleases:Early 1950s:observation –host-controlled restriction
Slide 16
Restriction occurs because the bacterium produces an enzyme that
degrades the phage DNA before it has time to replicate and direct
synthesisof new phage particles.
 Slide 17
Three different classes of restriction endonuclease:
---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.

type II restriction endonucleases: further referred only as restriction endonucleases have

recognition sequences at which it cuts a DNA molecule.

Example: PvuI (isolated from Proteus vulgaris) 5’ CGATCG 3’

Sau3A: GATC, and AluI (Arthrobacter luteus) cuts at AGCT.
restriction endonucleases with degenerate recognition sequences-
cut DNA at any one of a family of related sites: HinfI (Haemophilus
influenzae strain Rf recognizes GANTC, so cuts at GAATC, GATTC,
GAGTC, and GACTC
most frequently used restriction endonucleases Slide 18
Type II restriction endonucleases: Slide 19

---cut within the recognition site

--- recognition site is ds DNA and is written in 5’>3’ direction
---exact nature of the cut:
1. a simple double-stranded cut in the middle of the recognition sequence (Figure 4.9a),
resulting in a blunt end or flush end. PvuII and AluI are examples of blunt end cutters.

2. 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-sticky or cohesive ends
as base pairing between them can stick the DNA molecule back
together again
One important feature of sticky end enzymes is that restriction Slide 20
endonucleases with different recognition sequences may produce the same sticky
ends.
BamHI (recognition sequence GGATCC) and BglII (AGATCT) are examples—both
produce GATC sticky ends (Figure 4.9c). The same sticky end is also produced by
Sau3A, which recognizes only the tetranucleotide GATC

Pallindromic sequences