Nucleic Acid

DNA RNA

Nucleic Acids are the polymers of nucleotides.

Nucleotides are the combination of Nucleosides+ Phosphate

Nucleosides = Nitrogenous Base + Pentose Sugar

Nitrogen Base

Nitrogen Base

A G C T U

Purines Pyrimidines

+Deoxyribose pentose sugar

DNA

+Ribose Pentose Sugar

RNA

A T

G C

A= AdenineG = GuanineT = ThymineC = Cytosine

U = Urasil

Phosphate+

+ Phosphate

Duplex DNAH bond– DNA Strand<----- Polymer

Nucleotide

Nucleoside Phosphate

Nitrogen Base

A G C T /U

Pentose Sugar

Deoxyribose Ribose

What is Genetic Engineering???

Genetic engineering: The manipulation of genetic makeup of living cells by inserting desired gens through a DNA vector is known as genetic engineering.

Genetic Engineering involves:• removing a gene (target gene) from one organism• inserting target gene into DNA of another organism• ‘cut and paste’ process.

Gene : Small piece of DNA OR hereditary unit consisting of sequence of DNA

Alternative names for genetic engineering:

• Genetic Manipulation

• Genetic Modification

• Recombinant DNA Technology

• Gene Splicing

• Gene Cloning

Genetically Modified Organism (GMO): is the organism with the altered DNA.

This goat contains a human gene that codes for a blood clotting agent. The blood clotting agent can be harvested in the goat’s

milk.

How It Is Done???

1. Preparation Of Desired Gene2. Isolation of DNA vector3. Construction of Recombinant DNA (rDNA)4. Introduction of rDNA in to host cells5. Selection and multiplication of recombinant

host cells.6. Expression of cloned gene.

A Bacterial Cell

1. Preparation Of Desired Gene

Preparation Of Desired Gene

Using Restriction Enzymes

Using mRNA by Reverse

Transcriptase

Using m/c DNA Synthesizer

2. Isolation of DNA vector

• Vectors : The extrachromosomal DNA that carries desired gene to the host cell is called gene cloning vector.

• Eg. Plasmids, viral DNA, Cosmids etc

• Plamids : Plasmids are small, circular, double stranded extrachrosomal DNA present in bacterial cells.

3. Construction of Recombinant DNA (rDNA)

DNA 1 DNA 2

4.Introduction of rDNA in to host cells

1. Direct Transformation eg. Bacterial Cell intake rDNA

2. Pathological Agent eg. Bacteriophages & agarobacterium.

3. Liposomal Fusion eg. Animal/ Plant cells pick up rDNA in liposomes.

4. Direct Introduction eg. By microinjection or electron gun

5. Selection and screening of recombinant host cells

• Antibiotic resistance• Visible Characters• Assay of biological activity• Colony Hybridization

6. Expression of cloned gene.

• The desired gene expressed in the form of protein.

• The protein is isolated and tested immunologically.

Gene Clonning

• Gene clonning refers to in-vivo production of multiple copies of desired genes.

• In-vitro construction of rDNA and amplification of rDNA in bacterium or yeast.

• Inside the host cell the desired gene replicates along with the vector DNA by using replicative system and form more no of copies.

• As cell devides rDNA transferred to daughter cells.• Thus many identical copies of desired gene are

produced from a single rDNA.

Enzymes used for genetic engineering

• Restriction Endonucleases (DNA cutting Enzyme)• The enzyme that cut the DNA at a unique

sequence is called restriction endonuclease.• These are also known as molecular knives,

molecular scissors, restriction enzymes or molecular scalpels.

• Restriction site/ Recognition site.

Discovery

• In 1962, Werner Arber, a Swiss biochemist, provided the first evidence for the existence of "molecular scissors" that could cut DNA.

• Widespread among prokaryotes• He showed that E. coli bacteria have an

enzymatic “immune system” that recognizes and destroys foreign DNA, and modifies native DNA to prevent self-destruction.

Why don’t bacteria destroy their own DNA with their restriction enzymes?

Part I: Restriction

Bacteria produce restrictionenzymes that digest foreign (viral DNA)

Part II: Modification

Bacteria methylate their DNA toprotect it from digestion

Foreign DNA Host DNA

Types Of Restriction Enzymes

• Type I• Type II• Type III• Type I & Type III restriction enzymes recognize

specific sequence in duplex DNA but cut the DNA far away from the recognition sites. So they are not useful for genetic engineering.

• Type II restriction endonucleases recognize specific sites and cut the DNA at the recognized sites.

• Eg. ECoR I, Hind III etc• Molecular Weight – 20,000 to 1,00,000

daltons.• Naming….

Few Restriction Enzymes

Enzyme Organism from which derived

Target sequence

(cut at *)

5' -->3'

Bam HI Bacillus amyloliquefaciens G* G A T C C

Eco RI Escherichia coli RY 13 G* A A T T C

Hind III Haemophilus inflenzae Rd A* A G C T T

Mbo I Moraxella bovis *G A T C

Pst I Providencia stuartii C T G C A * G

Sma I Serratia marcescens C C C * G G G

Taq I Thermophilus aquaticus T * C G A

Xma I Xanthamonas malvacearum C * C C G G G

Mechanism Of Cutting

• Restriction Endonuclease scan the length of the DNA , binds to the DNA molecule when it recognizes a specific sequence and makes one cut in each of the sugar phosphate backbones of the double helix – by hydrolyzing the phoshphodiester bond. (5’ Phospahte group and 3’ OH group bonds)

– Covalent bonds (within a single strand)

– Hydrogen bonds (between strands) as a result of the strands coming apart

Hydrogen bond

Image taken without permission from http://www.bioteach.ubc.ca/MolecularBiology/RestrictionEndonucleases/endonuclease%202.gif

Covalent bond

What kinds of bonds are broken when restriction enzymes cut?

Based on the TYPES OF CUTS they make, there are two types

of restriction enzymes.

BLUNT ENDS

STICKY ENDS

5’... G A A T T C …3’3’... C T T A A G …5’

5’... G A A T T C …3’3’... C T T A A G …5’

Blunt Ends

Sticky Ends

Plane Of Cutting (Palindromic Sequence)

• Type II restriction enzymes recognizes a palindromic sequence to cut DNA.

Examples of Type II Restriction Endonucleases

Blunt ends

Cohesive“sticky” ends

Difference Between Type I & IIType I Restriction Endonucleases Type II R.E.

Mol wt. 400,000 Mol wt. 20,000 to 100,000 daltons

The enzyme has both endonuclease and methylase activity.

Restriction activity alone

The site of cutting is 1000 nucleotides away from the recognition site.

The site of cutting is the same recognition site

The sequence of cutting is non-specific The sequence of cutting is specific.

The enzymes protect DNAs by methylation. No methylation activity

ATP, Mg++ and adenosyl methionine are for activation. Mg++ alone required for activation

Uses

• Restriction enzymes are used to cut a source DNA into small frangments for clonning.

• Used to cut the unwanted sequence• Used to cut the vector DNA• Used to cut the larger DNA in to smaller

fragments.

DNA Ligase

• DNA ligase is an enzyme that joins the ends of two duplex DNA to make a long DNA. This process is known as ligation.

• It can’t add any nucleotide to a gap in the DNA.• Hydrogen bonds are not strong enough hence

phosphodiester bonds are formed.• 5’ Phosphate grp and 3’ OH grp forms

phosphodiester bond.

• DNA ligase is isolated from E-coli requires ATP and NAD+ for enzyme activity.

• However DNA ligase of lambda T4 phase requires ATP alone to catalyze the ligation.

• This enzyme is called T4 DNA ligase.• Mol wt. 68,000 daltons.

USES

• Used to join vector DNA and target DNA to construct rDNA

• Used to join DNA fragments of different organisms for making vectors.

• It is used to add linkers and adators sequence to blunt ended vector DNA and target DNA