RECOMBINANT

TECHNIQUES
Presented by Dr. Madhushree Pahari
2nd year PGT IPGME&R

MODERATOR
Prof(Dr.) Mousumi Mukhopadhyay
HOD,Dept.Of Biochemistry
IPGME&R and SSKMH
What is recombinant
technique?
•Techniques by which genetic
recombination is carried away in vitro.
•It entails breakage and rejoining of
DNA molecules from different
organisms and production and
isolation of modified DNA via artificial
means.
DISCOVERY
Goal of recombinant techniques

•Isolate and characterize a gene

•Artificially synthesize new gene
•Alternating genome of an organism
BASIC PRINCIPLES OF
RECOMBINANT DNA TECHNIQUE
• To produce DNA fragments using restriction
enzymes.
• Joining DNA fragments to other vector DNA
molecules by various techniques of ligation
• Cloning which is inserting resultant recombinant DNA
molecule into host cell where they self replicate to
produce multiple identical copies per cell of inserted
DNA molecule
PROCEDURES OF MAKING
RECOMBINANT DNA(rDNA)
• ISOLATING desired DNA
• CUTTING DNA with RESTRICTION ENZYME
• AMPLIFICATION with PCR
• SEPERATION of DNA BY GEL ELECTROPHORESIS
• LIGATION OF DNA MOLECULES
• INSERTING rDNA into host cell
• ISOLATION of recombinant cells
Tools used in recombinant
techniques
• ENZYMES :Restriction endonuclease,DNA ligase ,DNA
polymerase,alkaline phosphatase,reverse
transcriptase
• VECTOR:Plasmid,Bacteriophage,Cosmid,YAC
,BAC,MAC
• Host:prokaryotic>E coli,B
subtilis,Eukaryote>fungi :saccharomyces
cerevisiae,plant cell,mammalian cell
RESTRICTION
ENDONUCLEASE
• Bacteria use restriction enzymes to defend against
bacteriophages
• Restriction enzymes were named for their ability to restrict,
or limit, the number of strains of bacteriophage that can
infect a bacterium.
• HindII was the first restriction enzyme to be isolated. This
enzyme was first isolated from Haemophilus influenzae Rd
strain II.
RESTRICTION MODIFICATION
SYSTEM
• (Restriction endonuclease + methylase) is termed as
a restriction modification system, found in bacteria.
• A bacterium is immune to its own restriction enzymes,
even if it has the target sequences because the
bacterial restriction sites are highly methylated,
making them unrecognizable to the restriction
enzyme.
Restriction Modification SYSTEM
RESTRICTION
ENDONUCLEASE
• ENDONUCLEASE of prokaryotes also known as molecular scissor that
catalyzes the degradation of foreign DNA of a different bacteria or a
phage.
• Restriction enzyme recognise specific base sequences in DNA which
is generally palindromes, 4 -8 base pair in length known as
recognition or restriction site
• THREE MAJOR TYPES
• TYPE I AND TYPE III:recognise restriction site but type I make cut
1000 bp and type III make cut 25 bp away from recognition site
Type II restriction endonuclease
• Recognize restriction site in DNA and make double-stranded
cuts within the specific base sequence in DNA.
• These cuts based on restriction enzymes are of two types
A)Blunt End and B)Sticky End
• Blunt End:breaks occur at the centre of symmetry within
recognition site
• Sticky End:breaks occur equidistant from and on opposite
sides of the centre of symmetry within recognition site.
Sticky end ,blunt end

Sticky end Blunt end

• Ligation is efficient • Ligation is less efficient
• Could base pair with • Need high
themselves to form concentration of DNA
dimers and new DNA and ligase
molecule remain blunt
ended
STICKY END,BLUNT END
NUCLEIC ACID
LINKERS :treatment of blunt end
• Chemically synthesized double stranded DNA
oligonucleotides containing one or more restriction sites for
restriction enzymes :EcoRI,Hind III,Bam HI etc.
• Linkers are ligated to blunt end with DNA ligase
enzyme ,then it is digested with specific restriction enzymes
to produce sticky ends as ligation of blunt end DNA
fragments with cloning plasmid is not efficient as sticky end
DNA fragments
Treatment of blunt end
HOMOPOLYMER TAILING
• Sticky end can be produced on a blunt ended DNA
molecule
• Terminal deoxynucleotidyl transferase enzyme is used
to add homopolymer tail
• adds a series of nucleotides on the 3'-OH end of a
double-stranded DNA molecule.
• In a homopolymer all the subunits are same
ex;polydeoxyguanosine poly d(G)
Homopolymer tailing
CLONING VECTOR
• Vectors are used as carriers to deliver the desired
foreign DNA into a host cell
• All have at least one unique cloning site ,a sequence
that can be cut by a restriction endonuclease to allow
site specific insertion of foreign DNA
• Contain sequence that allow them to be replicated
autonomously within a compatible host
Plasmid
• Small circular extra chromosomal double stranded DNA molecule
• Can replicate independently
• Found in almost in all bacteria and some fungi,protozoa,Plants and
animals
Plasmid replication
PLASMID MAP
• Plasmid maps are graphical
representation of plasmids,
that show the locations of
three key parts
• Origin of replication
• Selectable marker gene
• Cloning site:restriction site
where DNA is inserted
A schematic representation of
the pBR322 vector with
restriction sites
Vectors:insert size
YAC:YEAST ARTIFICIAL
CHROMOSOME
• Genetically engineered DNA molecule
• Used to clone DNA sequences in yeast cell
• Mimics yeast chromosome
• Linear molecule composed of a centromere
telomeres,ARS(AUTONOMUS REPLICATING
SEQUENCE) and selectable markers
• Capable of replicating in common bacterial
host E.coli as well as in yeast s.cerevisiae
YAC:high
capacity
cloning
vector
vectors
• Bacteriophage:have the ability to replicate within the bacterial cells
and they provide the origin of replication
• Cosmid:hybrid plasmid that contains a Lambda phage cos sequence.
TRANSFECTION
• Transfection is the process of deliberately introducing purified nucleic acids
into eukaryote
• Some of the commonly used transfection techniques include calcium
phosphate precipitation, lipofection, electroporation, and viral delivery.
• Transfection can either be transient or stable.
• Transient transfection, the transfected material enters the cell but does not
get integrated into the cellular genome.
• Stable transfection, foreign DNA is either integrated into the cellular
genome or maintained as an extrachromosomal replicating chromosome.
TRANSFECTION

Stable transfection Transient transfection

ISOLATION OF DNA
ENZYMES USED IN DNA EXTRACTION
• LYSOZYME:lysis activity against gram negative
bacterial cell wall
• Achromopeptidase :lysis activity against gram positive
bacterial cell wall
• RNase A:degrade single stranded RNA
• Proteinase K:digestion of protein
ISOLATION OF DNA
Reagent used other than enzymes:
• SODIUM DODECYL SULFATE(SDS):solubilization cell
membrane lipid
• PHENOL:CHLOROFORM:ISOAMYL ALCOHOL SOLUTION(PCI
25:24:1):separation of DNA from other cellular
components
• Ethanol 100%:precipitate DNA from solution
• Tris EDTA buffer: used to store purified DNA
• Chelating agent EDTA:protects DNA from nuclease enymes
ISOLATION OF BACTERIAL DNA
BASIC STEPS
A)LYSIS:1)done by gentlest possible method in presence of Chelating
agent EDTA to prevent DNA fragmentation by inhibiting Dnase activity
2)Treated with detergent sodium dodecyl sulfate and
lysozyme,achromopepidase
B)ENZYME TREATMENT:homogenized cells are treated with a)RNase A
b)proteinase K for degradation of RNA and PROTEIN
C)CENTRIFUGATION: centrifugation of buffer lysed cell solution1)pellet
containing cell debris 2)supernatant containing intracellular
components
ISOLATION OF BACTERIAL DNA
D)PHENOL CHLOROFORM EXTRACTION:supernatant
treated with equal amount of phenol chloroform
isoamyl alcohol solution, centrifugation of mixture to
get supernatant aqueous layer with DNA molecules
E)ALCOHOL PRECIPITATION: done adding 70% to 100%
ethanol to aqueous DNA solution
F)REDISSOLVE DNA:Tris-EDTA buffer used to store
purified DNA
Isolation of
Bacterial DNA
SEPERATION OF DNA
• Gel electrophoresis is a technique used to separate DNA
fragments according to their size and charge.
• Principle:DNA samples are loaded into wells (indentations) at
one end of a gel, and an electric current is applied to pull them
through the gel. DNA fragments are negatively charged, so they
move towards the positive electrode.
• Agarose is appropriate for separating DNA fragments ranging in
size from a few hundred base pairs to large DNA FRAGMENTS
about 20 kb.
• Ethidium Bromide (EtBr) is sometimes added to running buffer
during the separation of DNA fragments by agarose gel
electrophoresis, EtBr binds to the DNA and allows it to visualize
the DNA under ultraviolet (UV) light.
Agarose gel electrophoresis
• Gels for DNA separation are made out of a polysaccharide called
agarose
• Agarose heated in a buffer and allowed to cool to prepare gel
• Buffers in gel electrophoresis are used to provide ions that carry a
current necessary for passage of electricity through gel and to
maintain the pH at a relatively constant value,buffer also keep gel
from melting.
• Tris/Acetate/EDTA (TAE) and Tris/Borate/EDTA (TBE) buffers used. TAE
has the lowest buffering capacity, but it provides the best resolution
for larger DNA.
AGAROSE GEL
ELECTROPHORESIS
DETECTION OF SPECIFIC DNA
SEQUENCE
• Southern blotting is a technique that allows the detection of a specific
region of DNA, The technique was named after its inventor, Edwin
Southern.
• BASIC STEPS OF SOUTHERN BLOT ANALYSIS:
• Step 1 DNA purification and RESTRICTION digestion.
• Step 2 Gel electrophoresis.
• Step 3 Denaturation with alkali solution.
• Step 4 Blotting.
• Step 5 Hybridization with labeled probe and washing.
• Step 6 Detection.
Southern blotting technique
Molecular Cloning
• Process Of creating clones of organism or copies of cell or DNA
fragments
Cloning of any DNA fragment essentially involves four steps
• fragmentation - breaking apart a strand of DNA
• ligation – gluing together pieces of DNA in a desired sequence
• transfection – inserting the newly formed pieces of DNA into
cells
• screening/selection – selecting out the cells that were
successfully transfected with the new DNA
Molecular cloning
REFERENCES
• Wilson and Walker’s principles and techniques of Biochemistry and
Molecular biology;8th edition
• Lehninger PRINCIPLES of BIOCHEMISTRY;7th edition
THANK YOU