Genetic engineering and

recombinant DNA
technology

Presented By:

Ashok Kumar (70700012)

Ila Chawla (70700019)
 What is Genetic engineering?

By artificial means, when a gene of one

species is transferred to another living
organism, it is called recombinant DNA
technology. In common parlance, this is
known as genetic engineering.
 Restriction enzymes

 Restriction enzymes allow DNA to be

cut at specific sites; Nucleic acid hybridization
allows the detection of specific nucleic acid
sequences; DNA sequencing can be used to
easily determine the nucleotide sequence of a
DNA molecule.
 Restriction endonuclease

Restriction endonuclease: recognize a short,

symmetrical DNA sequence, and cut DNA
backbone in each strand at a specific site
within that sequence
 Types of Restriction
endonuclease

Type I Type II Type III

Functions Endonucleas Endonucleae Endonuclease
e&
methylase
Conditions ATP, Mb2+ Mg2+ ATP, Mg2+

Recognition EcoK: Palindromic EcoP1: AGACC

sequences AACN6GTGC EcoP15:
EcoB: CAGCAG
TGAN8TGCT
Cutting sites At least At or close to 24-26 bp away
1000bp away recog. seq
 Recognition sequences

Recognize 4-8 bp palindromic sequences. Most commonly

used enzymes recognize 6 bp which occurs at a rate of
46=4096 bp. (44=256 bp; 48=65536 bp)

5’ GAATTC 3’
e.g. EcoRI site: 3’ CTTAAG 5’

Restriction enzymes
1. Highly specific
2. Commercially available
3. Require Mg2+ for enzymatic activity
4. Compatible ends from different enzymes,
 Restriction sequences

5’ protruding ends 3’ protruding ends

Cohesive/sticky ends
SmaI
5’-CCCGGG-3’ 5’-CCC-OH+ p -GGG-3’
3’-GGGCCC-5’ 3’-GGG- p OH-CCC-5’

blunt ends
 Vector

 The term “vector” here refers to some

DNA molecules that can carry a DNA
fragment into a host cell for replication.
 Including: plasmids, Bacteriophages
DNA, virus DNA ……
 Vectors used in molecular cloning

Vector Insert
(and host) Characteristics size range

Plasmid Small circular DNA <5 - 10 kb

(bacteria, yeast)
Bacteriophage λ Linear viral DNA up to ~20 kb
(bacteria)
Cosmid Hybrid of plasmid up to ~50 kb
(bacteria) and phage

Yeast artificial DNA containing yeast ~200 to

chromosome (YAC) centromere, telomeres, ~1000 kb
(yeast) and origins of replication
 Plasmid

• Plasmids are small, circular molecules of

DNA that exist outside the main bacterial
chromosome and carry their own genes for
specialized functions.
 Plasmid
　
 Agarose gel electrophoresis
Agarose: a polysaccharide derived from seaweed,
which
forms a solid gel when dissolved in aqueous solution
(0.5%-2%) Negatively charged DNA

- ve electrode + ve electrode
 Steps Involved in Gel Electrophoresis

1. “Cut” DNA sample with

restriction enzymes.

2. Run the DNA fragments

through a gel.

3. Bands will form in the gel.

4. Everyone’s DNA bands are

unique and can be used to
identify a person.

5. DNA bands are like “genetic

fingerprints”.
 Agarose gel electrophoresis

nicked

supercoiled
 Polymerase Chain Reaction(PCR)
　

 A technique used to make more copies of

 DNA in vitro (enzymatically)
 Requires all the building blocks of DNA
 DNA Polymerase (Taq polymerase)
 PCR reaction system
　
 DNA template
 A pair of primers
 DNA polymerase (Taq)
 dNTPs
 Mg2+-containing buffer
 Procedures of PCR
　
 Denaturing: the template DNA is
denatured to become ssDNA from dsDNA
by heating.
 Annealing: this step allows the
hybridization of the primers with target
DNA.
 Extension: this process is the DNA
synthesis step.
ing
 Process of cloning

 Isolation of target gene

 Selection and construction of vectors
 Ligation of target DNA and vector
 Transformation of target gene into
receptor cell
 Screening for recombinant plasmids
 Expressing a cloned gene 　
 Process of DNA cloning
　
 Isolation of target gene
　
1. Chemical synthesis
only for simple polypeptide chain whose
primary structure is clear.
2. Obtaining from genomic DNA library
3. Obtaining from cDNA library
4. polymerase chain reaction (PCR)
　 The genomic
DNA library is a
collection of the
comprehensive
DNA fragments
representing the
entire genome of a
species.
 mRNA
The cDNA library
　
represents the Reverse transcripase

population of cDNA
mRNAs, it only replication
contains the exons of
protein’s structural dscDNA
genes. vector

recombinate DNA
E. coli

recombinate DNA in E.coli

Selection and construction of vectors
　
A few commonly used vectors ：
plasmid
phage
cosmid
yeast artificial chromosome (YAC)
 Ligation of target DNA and vectors
　
1. Ligation of sticky end

GGATCC GGATCC
CCTAGG CCTAGG

G GATCC G GATCC
CCTAG G CCTAG G
DNA ligase
G GATCC
CCTAGG
　2. Ligation of blunt ends
3. The addition of a homopolymer tail
4.Artificial linker
　 Screening for recombinant

• Screen of antibiotic resistance markers

• Marker rescue (Insertion inactivation)
• In situ hybridization and autoradiography
 Antibiotic resistance genes
　
Screen of antibiotic resistance markers
In situ hybridization and autoradiography
　
　 Molecular markers

• Generally refers to the assays that allow

the detection of sequence differences
between two or more individuals.
　 There are 5 conditions that characterize
a suitable molecular marker:

 Must be polymorphic
 Co-dominant inheritance
 Randomly and frequently distributed throughout
the genome
 Easy and cheap to detect
 Reproducible
 　 Molecular markers can be used for
several different applications including:

 Germplasm characterization,
 Genetic diagnostics,
 Characterization of transformants,
 Study of genome
 Organization and phylogenic analysis.
TECHNIQUES USED FOR ANALYSIS OF
MOLECULAR MARKERS

 Restriction Digestion
 Gel Electrophoresis
 PCR
　 Marker- Type

1. Protein - based marker

2. DNA - based marker

　 Protein - based marker

Common protein marker: Isozymes

Multiple forms of the same enzyme

-allozyme: one enzyme and one locus

- isozyme: one enzyme, more than one locus

(gene duplication; gene families)

To be useful as markers, isoforms must be

electrophoretically resolvable, and detectable
by in-gel assay methods
　 DNA -based marker

Advantages
– not influenced by environment
– expressed in all tissues

RFLPs - restriction fragment length polymorphisms

PCR-based markers
RAPDs
SSRs
AFLPs
 Molecular Marker Techniques

Restriction Fragment Length Polymorphism (RFLP)

 The technique centers around the digestion of genomic DNA
digested with restriction enzymes.

 These enzymes are isolated from bacteria and consistently cut DNA
at specific base pair sequences which are called recognition sites.

 These recognition sites are not associated with any type of gene
and are distributed randomly throughout the genome.

 When genomic DNA is digested with one of these restriction

enzymes, (of which there are thousands, each cutting at a specific
sequence), a series of fragment are produced of varying length.
 These fragments are separated using agarose or
polyacrylamide gel electrophoresis (PAGE) and
yield a characteristic pattern.

 Variations in the characteristic pattern of a RFLP

digest can be caused by
 base pair deletions,
 mutations,
 inversions,
 translocations and transpositions
 which result in the loss or gain of a recognition
site resulting in a fragment of different length and
polymorphism.
 RFLP

Enzymes cut DNA at specific sequences

Restriction sites are often palindromes:

6-cutter GAATTC 4-cutter TCGA

CTTAAGAGCT
 Using RFLP polymorphism

Advantages:

 variants are co-dominant;

 measures variation at the level of DNA sequence, not
protein sequence.

Disadvantages:

 labor intensive;
 requires relatively large amounts of DNA
 PCR Based Molecular Markers

Randomly amplified polymorphic DNA Markers (RAPD)

 RAPD was the first PCR based molecular marker technique
developed and it is by far the simplest.

 Short PCR primers (approximately 10 bases) are randomly and

arbitrarily selected to amplify random DNA segments throughout the
genome.

 The resulting amplification product is generated at the region

flanking a part of the 10 bp priming sites in the appropriate
orientation.

 RAPD often shows a dominant relationship due to primer being

unable to bind on recessive alleles.

 RAPD products are usually visualized on agarose gels stained with

ethidium bromide.
RAPD: Randomly amplified polymorphic DNA

Size sorted
RAPDs
Advantages:
 fast,
 relatively inexpensive,
 highly variable.

Disadvantages:
 markers are dominant.
 Presence of a band could mean the individual is either
heterozygous or homozygous for the sequence--can’t tell
which.
 Data analysis more complicated.
Amplified Fragment Length Polymorphism (AFLP)

 AFLP is the latest form of marker assisted selection and

is a highly sensitive method based on the combined
concepts of RFLP and RAPD.

 This technique is applicable to all species giving very

reproducible results.

 The basis of AFLP is the PCR amplification of restriction

enzyme fragments of genomic DNA.
AFLPs

Advantages:

 fast,
 relatively inexpensive,
 highly variable.

Disadvantages:

 markers are dominant.

 Presence of a band could mean the individual is either
heterozygous or homozygous for the sequence--can’t tell
which.