Genetic Engineering

Recombinant DNA Technology

Deepak Sehgal

Cell Biology : An Introduction

All living organisms are made

out of cells
Cells are the smallest living

unit
Cell is the structural and

functional unit of life.

Human egg cell + sperm

Cell Types

http://library.thinkquest.org/12413/structures.html

DNA

DNA Functions
Stores genetic

information to build and maintain a living organism Copies itself

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James Watson & Francis Creek 1953

Determined the three-

dimensional structure of DNA One of the greatest discoveries in the history of science Nobel prize in 1962
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DNA
Made of repeating units called nucleotides Each nucleotide made of a sugar named deoxyribose, a phosphate and a base

DNA Nucleotide
Deoxyribose Base

Phosphate

DNA Molecule is Double Stranded

Base Pairing Rule

Adenine pairs with

Thymine Guanine pairs with Cytosine

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DNA
Total length of DNA in each

cell is about 6 feet Wraps around proteins called histones Over 3 billion nucleotide pairs in human DNA!
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Each new DNA molecule will be an exact copy of the original

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DNA is the genetic material of most of the organisms: from Bacteria to Humans
Plasmid

Chromosome: Most bacteria have one circular DNA chromosome

ranging in size from 1,000 to 8,000 kilobase pairs. Plasmid: Extrachromosomal genetic element also made of a circular DNA molecule. Bacterial Genome: The collection of all of the genes present on the bacterias chromosome or its extrachromosomal genetic elements.

DNA Double Helix

Rungs of ladder Nitrogenous Base (A,T,G or C)

Legs of ladder

Phosphate & Sugar Backbone

DNA Nucleotide
Phosphate Group

O O=P-O O

CH2 O N

C4
Sugar (deoxyribose)

C1

Nitrogenous base (A, G, C, or T)

C3

C2

BASE-PAIRINGS
H-bonds

What is recombinant DNA?

Recombinant DNA is an

artificial strand of DNA created by combining DNA segments from two or more sources.
It is the basis for all sorts of

genetic engineering.

GM fluorescent plant
Science Magazine

Uses of recombinant DNA

Genetically modified crops

papilio.ab.a.utokyo.ac.jp

(Roundup Ready) Genetically modified animals for research (Oncomouse) Genetically modified microorganisms to produce pharmaceuticals (insulin) Gene therapy (Cystic Fibrosis) To isolate and study a particular gene (subcloning) Creating transgenic organisms or chimeras.

Transgenic insect

What Does It Mean: To Clone?

Clone: a collection of molecules or cells, all identical to an original molecule or cell
To "clone a gene" is to make many copies of it - for example, by

replicating it in a culture of bacteria.

Cloned gene can be a normal copy of a gene (= wild type). Cloned gene can be an altered version of a gene (= mutant). Recombinant DNA technology makes manipulating genes possible.

 What are the 3 general steps used to clone DNA?

Isolate DNA from an organism Cut the organismal DNA and the vector with restriction enzymes

making recombinant DNA Introduce the recombinant DNA into a host

Restriction Enzymes
Recognize a specific site (generally a pallidromic sequence) Produce overhangs or straight cuts Naturally found in bacteria, they protect against viruses and

foreign DNA More than 400 enzymes have been isolated Named for the organism they from which they are isolated The first letter is that of the genus and the 2nd and 3rd are from the species

Restriction Enzymes
Bacteria have learned to "restrict" the possibility of attack from

foreign DNA by means of "restriction enzymes.

Cut up foreign DNA that invades the cell. Enzymes may recognize 4, 6 or more bases in selecting sites for

cleavage.
An enzyme that recognizes a 6-base sequence is called a "six-

base cutter.

Recognition/Cleavage Sites of Type II Restriction Enzymes

Cuts usually occurs at a palindromic sequence SmaI: produces blunt ends 5 CCCGGG 3 3 GGGCCC 5

EcoRI: produces sticky ends 5 GAATTC 3 3 CTTAAG 5

DNA cloning requires restriction endonuclease and DNA ligase

Consider a plasmid with a unique EcoRI site: 5' NNNNGAATTCNNNN 3' 3NNNNCTTAAGNNNN 5' An EcoRI restriction fragment of foreign DNA can be inserted into a plasmid having an EcoRI cloning site by: a) cutting the plasmid at this site with EcoRI, b) annealing the linearized plasmid with the EcoRI foreign DNA fragment, and, c) sealing the nicks with DNA ligase. 5' NNNNGAATTCNNNN 3' 3' NNNNCTTAAGNNNN 5 This results in a recombinant DNA molecule.

Restriction Endonucleases Cleave DNA at specific DNA sequences

Central Dogma of Genetics: DNA RNA Protein

DNA
Transcription
RNA Processing

Nuclear membrane

Pre-mRNA

mRNA Ribosome
Translation

Protein Eukaryotic Cell

A mixture of dNTPs and ddNTPs are used in DNA sequencing

Polyacrylamide gel electrophoresis is used to visualize the results of the sequencing reaction

Automated DNA sequencing output4 reactions carried out in one tube

Polymerase Chain Reaction (PCR)

Allows quick identification of genetic markers:
Identify bacteria in infections Identify viruses in virus infections Paternity testing, genetic counseling, forensics Can exclude individuals, but cannot prove guilt.

Requires only small amounts of DNA. A repetitive DNA synthesis reaction.

Thermostable DNA polymerase: Isolated from bacteria in hot springs

or near thermal vents in the deep ocean.

Requires gene-specific DNA primers and deoxyribonucleotide triphosphates (dNTPs).

A thermophilic (heat-loving) bacteria called Thermus aquaticus is the source of Taq DNA polymerase used in PCR reactions

The first round of PCR

94C 37-65C

70-75C

PCR increases the yield of DNA exponentially

PCR in Medicine
Since 1987, PCR has had a major impact on prenatal diagnosis of single gene disorders.

Also very important in carrier testing for genetic diseases.

Improved speed, accuracy and technical flexibility over previous methods.

PCR and prenatal diagnosis

For prenatal diagnosis, PCR used to amplify DNA from fetal cells obtained from amniotic fluid. Single base changes then detected by one or more of following: -dot blot (spot hybridization) with oligonucleotides specific for known mutation. -restriction enzyme analysis (RFLP). -direct sequencing of DNA.

Important to be certain of result so combination of two methods provides confirmation.

Many other conditions can be detected with same approach, including: Tay-Sachs disease, phenylketonurea, cystic fibrosis, hemophilia, Huntingdon's disease, Duchenne muscular dystrophy (DMD).

PCR to detect HIV

PCR allows the direct detection of HIV genomes in patient blood before the appearance of HIV antibodies.
viral DNA/RNA only represents a minute proportion of total cell DNA. Only a small fraction of blood cells are infected (1/10,000). also require high degree of specificity while targeting conserved regions of DNA to guard against high level of genetic variability characteristic of retroviruses.

High risk of cross-contaminating sample with small amounts of amplified DNA from previous sample requires extra precautions to prevent false-positives.
PCR can detect 10-20 copies of viral DNA from 150,000 human cells.

PCR in Forensics
Crucial forensic evidence may be present in very small quantities. often too little material for direct DNA analysis. but PCR can generate sufficient DNA from a single cell. PCR also possible on extensively degraded DNA. examples include DNA from single dried blood spot, saliva (on cigarette butt), semen, tissue from under fingernails, hair roots. Other advantages of PCR in forensic science are: relatively simple to perform and simple to standardize. results obtainable within 24 hours.

Central Dogma of Genetics: DNA RNA Protein

DNA
Transcription
RNA Processing

Nuclear membrane

Pre-mRNA

mRNA Ribosome
Translation

Protein Eukaryotic Cell

 1. Sehgal, D and K.P.Gopinathan (1998) Overexpression of Green fluorescent

Protein in Bombyx mori using baculovirus expression vector system. Biotechniques 25: 997-1007.

2. Sehgal, D., Malik, P. S. and Jameel, S. (2003) Purification and diagnostic utility of the hepatitis E virus capsid protein expressed in insect larvae. Protein Expr. Purif. 27: 27-34.

Thank You !

Confidential

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