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Genetic engineering and

recombinant DNAtechnology

Presented By:

Ashok Kumar (70700012)

Ila Chawla (70700019)

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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.

What is Genetic engineering?

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Restriction enzymes allow DNA to be

cut at specific sites; Nucleic acid hybridization

allows the detection of specific nucleic acidsequences; DNA sequencing can be used to

easily determine the nucleotide sequence of a

DNA molecule.

Restriction enzymes

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Restriction endonuclease: recognize a short,

symmetrical DNA sequence, and cut DNA

backbone in each strand at a specific sitewithin that sequence

Restriction endonuclease

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Types ofRestriction endonuclease

Type I Type II Type III

Functions Endonucleas

e &

methylase

Endonucleae Endonuclease

Conditions ATP, Mb2+ Mg2+ ATP, Mg2+

Recognition

sequences

EcoK:

AACN6GTGC

EcoB:

TGAN8TGCT

Palindromic EcoP1: AGACC

EcoP15:

CAGCAG

Cutting sites At least

1000bp away

At or close to

recog. seq

24-26 bp away

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Restriction enzymes

Recognize 4-8 bp palindromic sequences. Most commonlyused enzymes recognize 6 bp which occurs at a rate of

46=4096 bp. (44=256 bp; 48=65536 bp)

1. Highly specific

2. Commercially available

3. Require Mg2+ for enzymatic activity

4. Compatible ends from different enzymes,

5 GAATTC 33 CTTAAG 5e.g. EcoRI site:

Recognition sequences

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5 protruding ends 3 protruding ends

5-CCCGGG-3

3-GGGCCC-55-CCC-OH

3-GGG- p

p -GGG-3

OH-CCC-5+

SmaI

blunt ends

Cohesive/sticky ends

Restriction sequences

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Vector

The term vector here refers to some

DNA molecules that can carry a DNAfragment into a host cell for replication.

Including: plasmids, Bacteriophages

DNA, virus DNA

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Vectors used in molecular cloning

Vector Insert(and host) Characteristics size range

Plasmid Small circular DNA

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Plasmid

Plasmids are small, circular molecules of

DNA that exist outside the main bacterial

chromosome and carry their own genes for

specialized functions.

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Plasmid

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Agarose: a polysaccharide derived from seaweed, which

forms a solid gel when dissolved in aqueous solution

(0.5%-2%)

- ve electrode + ve electrode

Negatively charged DNA

Agarose gel electrophoresis

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Steps Involved in Gel Electrophoresis

1. Cut DNA sample withrestriction enzymes.

2. Run the DNA fragments

through a gel.

3. Bands will form in the gel.

4. Everyones DNA bands are

unique and can be used toidentify a person.

5. DNA bands are like geneticfingerprints.

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supercoiled

nicked

Agarose gel electrophoresis

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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)

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PCR reaction system

DNA template

A pair of primers

DNA polymerase (Taq) dNTPs

Mg2+-containing buffer

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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 DNAsynthesis step.

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ing

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Process of DNA cloning

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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)

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The genomic

DNA library is acollection of the

comprehensive

DNA fragmentsrepresenting the

entire genome of a

species.

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The cDNA library

represents thepopulation of

mRNAs, it only

contains the exons ofproteins structural

genes.

mRNA

Reverse transcripase

cDNA

replication

dscDNA

vector

recombinate DNAE. coli

recombinate DNA in E.coli

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A few commonly used vectors

plasmid

phage

cosmid

yeast artificial chromosome (YAC)

Selection and construction of vectors

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GGATCC

CCTAGG

GGATCC

CCTAGG

G

CCT

AG

GATCC

G

G

CCT

AG

GATCC

GA li

G

CCTAG

GATCC

G

Li tion of t r t A nd v ctor

1. Ligation of sticky end

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2. Ligation of blunt ends

3. The addition of a homopolymer tail

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Screening for recombinant

Screen of antibiotic resistance markers

Marker rescue (Insertion inactivation)

In situ hybridization and autoradiography

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Antibiotic resistance genes

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Screen of antibiotic resistance markers

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In situ hybridization and autoradiography

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Molecular markers

Generally refers to the assays that allow

the detection of sequence differences

between two or more individuals.

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Must be polymorphic

Co-dominant inheritance

Randomly and frequently distributed throughoutthe genome

Easy and cheap to detect

Reproducible

There are 5 conditions that characterize

a suitable molecular marker:

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Germplasm characterization,

Genetic diagnostics,

Characterization of transformants,

Study of genome

Organization and phylogenic analysis.

Molecular markers can be used for

several different applications including:

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TECHNIQUES USED FOR ANALYSIS OF

MOLECULAR MARKERS

Restriction Digestion

Gel Electrophoresis

PCR

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Marker- Type

1. Protein - based marker

2. DNA - based marker

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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

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DNA -based marker

Advantages

not influenced by environment

expressed in all tissues

RFLPs - restriction fragment length polymorphisms

PCR-based markers

RAPDs

SSRs

AFLPs

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Molecular MarkerTechniques

Restriction Fragment Length Polymorphism (RFLP)

The technique centers around the digestion of genomic DNAdigested with restriction enzymes.

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

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

When genomic DNA is digested with one of these restrictionenzymes, (of which there are thousands, each cutting at a specificsequence), a series of fragment are produced of varying length.

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These fragments are separated using agarose orpolyacrylamide gel electrophoresis (PAGE) andyield 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 andpolymorphism.

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RFLP

6-cutterGAATTC 4-cutter TCGACTTAAG AGCT

Enzymes cut DNA at specific sequences

Restriction sites are often palindromes:

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Advantages:

variants are co-dominant;

measures variation at the level of DNA sequence, notprotein sequence.

Disadvantages:

labor intensive;

requires relatively large amounts of DNA

Using RFLP polymorphism

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PCR Based Molecular Markers

Randomly amplified polymorphic DNA Markers (RAPD)

RAPD was the first PCR based molecular marker techniquedeveloped and it is by far the simplest.

Short PCR primers (approximately 10 bases) are randomly andarbitrarily selected to amplify random DNA segments throughout thegenome.

The resulting amplification product is generated at the regionflanking a part of the 10 bp priming sites in the appropriateorientation.

RAPD often shows a dominant relationship due to primer beingunable to bind on recessive alleles.

RAPD products are usually visualized on agarose gels stained withethidium bromide.

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RAPD:

Randomly amplified polymorphic DNA

Size sorted

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RAPDs

Advantages: fast,

relatively inexpensive,

highly variable.

Disadvantages:

markers are dominant.

Presence of a band could mean the individual is eitherheterozygous or homozygous for the sequence--cant tellwhich.

Data analysis more complicated.

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Amplified Fragment Length Polymorphism (AFLP)

AFLP is the latest form of marker assisted selection andis a highly sensitive method based on the combinedconcepts of RFLP and RAPD.

This technique is applicable to all species giving veryreproducible results.

The basis of AFLP is the PCR amplification of restrictionenzyme fragments of genomic DNA.

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AFLPs

Advantages:

fast,

relatively inexpensive,

highly variable.

Disadvantages:

markers are dominant.

Presence of a band could mean the individual is eitherheterozygous or homozygous for the sequence--cant tellwhich.

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