Lecture 2 Recombinant DNA - PowerPoint Presentation

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Lecture

2Recombinant DNA Technology and Biotechnology

FE314

-

Biotechnology

Spring

201

7

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Technique of manipulating the genome of a cell or organism so as to change the phenotype desirably.

What is recombinant DNA ?

Seedless guava

Calorie free sugar

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DNA structure

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#1. DNA Structure (an overview)

DNA has three main components1. deoxyribose (a pentose sugar)2. base (there are four different ones)3. phosphate

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#2. The Bases

They are divided into two groupsPyrimidines and purines

Pyrimidines (made of one 6 member ring)ThymineCytosine

Purines

(made of a 6 member ring, fused to a 5 member ring)

Adenine

Guanine

The rings are not only made of carbon (specific formulas and structures are not required for IB)

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#3. Nucleotide Structure

Nucleotides are formed by the condensation of a pentose sugar, phosphate and one of the 4 basesThe following illustration represents one nucleotide

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Nucleotides are linked together by covalent bonds called phosphodiester linkage

#3. Nucleotide Structure

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#4. DNA Double Helix and Hydrogen Bonding

Made of two strands of nucleotides that are joined together by hydrogen bondingHydrogen bonding occurs as a result of complimentary base pairingAdenine and thymine pair upCytosine and guanine pair upEach pair is connected through hydrogen bonding

Hydrogen bonding always occurs between one pyrimidine and one purine

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Complimentary base pairing of pyrimidines and purines

#4. DNA Double Helix and Hydrogen Bonding

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#4. DNA Double Helix and Hydrogen Bonding

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#1. DNA Structure (an overview)

DNA has three main components1. deoxyribose (a pentose sugar)2. base (there are four different ones)3. phosphate

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#2. The Bases

They are divided into two groupsPyrimidines and purines

Pyrimidines (made of one 6 member ring)ThymineCytosine

Purines

(made of a 6 member ring, fused to a 5 member ring)

Adenine

Guanine

The rings are not only made of carbon (specific formulas and structures are not required for IB)

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#3. Nucleotide Structure

Nucleotides are formed by the condensation of a pentose sugar, phosphate and one of the 4 basesThe following illustration represents one nucleotide

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Nucleotides are linked together by covalent bonds called phosphodiester linkage

#3. Nucleotide Structure

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#4. DNA Double Helix and Hydrogen Bonding

Made of two strands of nucleotides that are joined together by hydrogen bondingHydrogen bonding occurs as a result of complimentary base pairingAdenine and thymine pair upCytosine and guanine pair upEach pair is connected through hydrogen bonding

Hydrogen bonding always occurs between one pyrimidine and one purine

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Complimentary base pairing of pyrimidines and purines

#4. DNA Double Helix and Hydrogen Bonding

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#4. DNA Double Helix and Hydrogen Bonding

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Adenine always pairs with thymine because they form two H bonds with each other

Cytosine always pairs with guanine because they form three hydrogen bonds with each other

#4. DNA Double Helix and Hydrogen Bonding

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The ‘backbones’ of DNA molecules are made of alternating sugar and phosphates

The ‘rungs on the ladder’ are made of bases that are hydrogen bonded to each other#5. DNA Double Helix

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#6. Antiparallel strands

The strands run opposite of each other.

The 5’ end always has the phosphate attached.

5’

3’

3’

5’

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Assignment (in your notebook)

1. Draw the structure of ribose and number the carbons2. Draw a schematic representation of a nucleotide. Label the sugar, base and phosphate.3. What are the complimentary base pairs to a DNA strand that has the following order A T A C C T G A A T?4. Draw a schematic representation of an unwound DNA double helix using the base pairs from your answer in question 3.

Include the number of hydrogen bonds between each base pair. Be sure to label all of the bases and the 5’ and 3’ ends of the structure.

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#6. When phosphodiester links are formed . . .

A. When the covalent bonds are formed between nucleotides the attach in the direction of 5’→3’B. The 5’ end of one nucleotide attaches to the 3’ end of the previous nucleotide

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#7. Nucleosome structure

Nucleosome are the basic unit of chromatin organizationIn eukaryotes DNA is associated with proteins(in prokaryotes the DNA is naked)Nucleosomes = basic beadlike unit of DNA packingMade of a segment of DNA wound around a protein core that is composed of 2 copies of each of 4 types of histones

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#8. Genes

Genes=units of genetic information (hereditary information)Order of nucleotides make up the genetic codeGenes can contain the information for one polypeptideGenes can also regulate how other genes are expressedAll cells of an organism contain the same genetic information but they do not all express the same genes

THIS IS CELL DIFFERENTIATIONCells differentiate by genes that are activated

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Repetitive sequences-part of the non-coding section of DNA

Function-unknownCan be used in DNA profiling (DNA fingerprinting)#8. Genes

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Basic steps involved in process

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Basic steps involved in process

Isolating genomic DNA

1.

Isolating genomic DNA from the donor.

Fragmenting this DNA

2.

Fragmenting this DNA using molecular scissors.

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Basic steps involved in process

Insertion of DNA in a vector

3.

Screening the fragments

4.

Screening the fragments for a “desired gene”.

Inserting the fragments with the desired gene in a ‘cloning vector’.

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Basic steps involved in process

Introducing in Host

Culturing the cells

Transformation of host cell

Introducing the recombinant vector into a competent host cell

Culturing these cells to obtain multiple copies or clones of desired DNA fragments

Using these copies to transform suitable host cells so as to express the desired gene.

5.

6.

7.

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Tools used in recombinant DNA technology

Enzymes Vectors

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Tools used in recombinant DNA technology

EnzymesAct as biological scissors.Most commonly used are: Restriction endonuclease

DNA ligaseDNA polymeraseAlkaline phosphatases

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Tools used in recombinant DNA technology

VectorsLow molecular weight DNA molecules.Transfer genetic material into another cell.Capable of multiplying independently.

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Vector

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Insertion of vector in target cell is called

Bacterial cells – TransformationEukaryotic cells – TransfectionViruses - Transduction

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Insertion of vector in target cell

Vectors used:Bacteria- plasmids, cosmid, lambda phageInsects- baculovirusesPlants- Ti plasmidYeast cells- YAC (yeast artificial chromosome)

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Process

HOST

DONORDNA

Fragmented by Restriction

Endonuclease

DNA strands with sticky ends

Sticky ends base pair with complementary sticky ends

DNA

ligase

links them to form

rDNA

Cloned

In vivo

In vitro

Prokaryotic or eukaryotic cell, mammalian tissue culture cell

DNA

Polymerase chain reaction (

PCR)

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Some examples of therapeutic products made by recombinant DNA techniques

Blood Proteins: Erythropoietin, Factors VII, VIII, IX; Tissue plasminogen activator; Urokinase.Human Hormones: Epidermal growth factor; Follicle stimulating hormone, Insulin.

Immune Modulators: α Interferon, β Interferon; Colony stimulating hormone; Lysozyme; Tumor Necrosis factor.Vaccines: Cytomegalovirus; Hepatitis B; Measles; Rabies

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Transposons

Transposons are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell.Also called ‘Jumping genes’.

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1st

transposons were discovered by

Barbara McClintockin Zea mays (maize)

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Types of transposons

According to their mechanism they are classified as:

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Retrotransposons

Follows method of “Copy and Paste”.Copy in two stages.

DNA

DNA

RNA

Reverse Transcription

Transcription

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DNA transposons

Follows the method of “Cut and Paste”.Do not involve RNA intermediate.Enzyme Transposase

Cuts out transposonLigates in new position

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Plasmid

Plasmids are small, extra chromosomal, double stranded, circular forms of DNA that replicate autonomously.The term was introduced by in 1952.

Joshua Lederberg

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Plasmid

Found in bacterial, yeast and occasionally in plants and animal cells. Transferable genetic elements or ‘Replicons’.Size- 1 to 1000 kilo bp.Related to metabolic activity.Allows bacteria to reproduce under unfavorable conditions.

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Nomenclature

Lower case P (p)First letters of researchers name or place where it was discovered.Numerical numbers given by workers. Plasmid

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Plasmid

Eg. Plasmid pBR 322

BR is for Bolivar and Rodriguez, who designated it as 322

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Plasmid

Eg. Plasmid pUC 19

UC stands for University of California

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Plasmid- Cosmids

Cosmids are plasmids with cos sequence.They are able to accommodate long DNA fragments that plasmids can’t.

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A bacteriophage is a virus that infects bacteria.Virulent portion is deleted.

Bacteriphages

Genetic material can be ssRNA, dsRNA,

ssDNA

,

dsDNA

.

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For Single genes- Plasmids are used

For Large pieces of DNA- BacteriophagesVectors used

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48.5 kb in length.Cos sites of 12 bp at the ends.Cohesive ends allow circularizing DNA in host.

Phage Lambda () as vector

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Lytic Cycle (replication of bacteriophges)

Phage attaches to a specific host bacterium.

Injects its DNA, Disrupting the bacterial genome and killing the bacterium, and Taking over the bacterial DNA and protein synthesis machinery to make phage parts. The process culminates with the assembly of new phage, andThe lysis of the bacterial cell wall to release a hundred new copies of the input phage into the environment.

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Restriction fragments

A restriction fragment is a DNA fragment resulting from the cutting of a DNA strand by the restriction enzyme. Process is called restriction.

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Restriction fragments

Steward Linn along with Werner Arber in 1963 isolated two enzymes.One of them is Restriction Endonuclease. Restriction Endonuclease can cut DNA.Restriction Endonuclease are basic requirement for gene cloning or rDNA technology.

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Restriction fragments

They remove nucleotides from the ends of the DNA

They make cuts at specific positions within the DNA

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Types of REN

Mostly used in

rDNA technology.More than 350 types of type II endonucleases with recognition sites are known.Can be used to identify and cleave within specific DNA.

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Nomenclature of ren

First letter- genus name of bacteria (in italics).Next- first two letters of the species name (in italics).Next- strain of the organism.Roman number- order of discovery.

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Eg. -

EcoR IE- Escherichia, co- coli, R-strain Ry 13,

I- first endonuclease to be discovered.Eg.- Hind IIIH-

Haemophilus

, in-

influenzae

, d- strain Rd,

III- third

endonuclease

to be discovered.

Nomenclature of

ren

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Recognition sequence (restriction sites)

It is the site/ sequence where REN cuts the DNA.Sequence of 4-8 nucleotides.Most restriction sites are Palindromes.

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In DNA, palindrome is a sequence of base pairs that reads the same on the two strands when orientation of reading is kept same.

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Cleavage patterns of ren

REN recognizes the restriction site.Cleave the DNA by hydrolyzing Phosphodiester bonds.Isolate a particular gene.Single stranded ends called sticky ends.

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These sticky ends can form hydrogen bonded base pairs with complementary sticky ends or any other cleaved DNA.

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Cleavage patterns of ren

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Restriction fragments yield a band pattern characteristic of the original DNA molecule & restriction enzyme used.

Restriction fragments can be analyzed by

Gel electrophoresis

Bands

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Preparing and cloning a DNA library

Collection of DNA fragments from a particular species that is stored and propagated in a population of micro organisms through molecular cloning.

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Genomic library

Collection of all clones of DNA fragments of complete genome of an organism.All DNA fragments are cloned and stored as location of desired gene is not known.Screening of DNA fragments can be done by Complementation Or by using Probes.

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Construction of Genomic Library.

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Cdna library

cDNA is Complementary DNA.Produced using Teminism i.e. Reverse Transcriptase.Constructed for eukaryotes.

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cDNA is made from mRNA

Mature mRNA

Start

AAAAAAA

Stop

TTTTTTT

Add

polyT

primer, nucleotides, and Reverse Transcriptase

TTTTTTT

AAAAAAA

TTTTTTT

DNA/RNA

RNA removed (by

NaOH

) and second strand synthesized

Complementary DNA cDNA

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Gene Amplification (PCR)

It is obtaining multiple copies of a known DNA sequences that contain a gene.Done artificially by using PCR (Polymerase Chain Reaction)

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PCR (Polymerase Chain Reaction)

Developed by in 1983.In Vitro technique.Scientific technique to generate billions of copies of a particular DNA sequence in a short time.

Kary Mullis

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PCR Machine

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Requirements for PCR technique

A DNA segment 100-35,000 bp in length to be amplified.

Primers-forward and reverse, are synthetic oligonucleotides and complementary to the desired DNA segmentFour types of deoxyribonucleotides i.e. dCTP, dGTP

,

dTTP

,

dATP

Enzyme that can withstand

upto

94° C.

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Steps of PCR technique

 The double strand melts open to single stranded DNA, all enzymatic reactions stop (for example : the extension from a previous cycle).

 Ionic bonds are constantly formed and broken between the single stranded primer and the single stranded template. Once there are a few bases built in, the ionic bond is so strong between the template and the primer, that it does not break anymore.The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's

from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template)

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The exponential amplification of the gene in PCR.