DNA TECHNOLOGYDNA recombination or genetic

engineering is the direct manipulation of genes for practical purposes

Recombinant DNA technology• Refers to the set of techniques for

combining genes from different sources in vitro( in a test tube) and transfering this DNA into a cell so it can be expressed.

• These techniques were first developed around 1975 and resulted in the appearance of the Biotechnology industry

•12.7 DNA technology is changing the pharmaceutical industry

– DNA technology• Is widely used to produce medicines and to

diagnose diseases

CONNECTION

What is biotechnology?• The use of living organisms to do practical

tasks.

• Examples:

• The use of microorganisms to make cheese and wine

• Selective breeding of livestock and crops

• Production of antibiotics from microorganisms

• Production of monoclonal antibodies

What is the goal of biotechnology?

• To find practical applications of DNA tecniques for the improvement of human health and food production

• Examples:

Making gene products using Genetic Engineering

Uses in basic research

Medical uses. Diagnosis of disease

Making vaccines and other pharmaceutical products

Forensic uses of DNA such as DNA fingerprinting

Agricultural uses such as manipulatingplant genes and making transgenic plants.

The tools of recombinant DNA

• Plasmids

• Restriction enzymes

• Gel electrophoresis

• PCR ( polymerase chain reaction)

BACTERIAL PLASMIDS AND GENE CLONING

•12.1 Plasmids are used to customize bacteria: An overview

– Gene cloning is one application of DNA technology

• Methods for studying and manipulating genetic material

– Researchers can insert desired genes into plasmids, creating recombinant DNA

• And insert those plasmids into bacteria

Bacterium

Bacterialchromosome

Plasmid

1 Plasmidisolated

3 Gene insertedinto plasmid

2 DNAisolated

Cell containing geneof interest

DNAGene ofinterest

Recombinant DNA(plasmid)

4 Plasmid put intobacterial cell

Recombinantbacterium

5 Cell multiplies withgene of interest

Copies of proteinCopies of gene

Clone of cellsGene for pestresistanceinserted intoplants

Gene used to alter bacteriafor cleaning up toxic waste

Protein used to dissolve bloodclots in heart attack therapy

Protein used tomake snow format highertemperature

Figure 12.1

•Therapeutic hormones– In 1982, humulin, human insulin produced by

bacteria• Became the first recombinant drug approved

by the Food and Drug Administration

Figure 12.7A

– Gene therapy• Is the alteration of an afflicted individual’s genes

Gene therapy may someday help treat a variety of diseases

Cloned gene(normal allele) 1 Insert normal gene

into virus

2 Infect bone marrowcell with virus

3 Viral DNA insertsinto chromosome

4 Inject cellsinto patient

Bonemarrow

Bone marrowcell from patient

Viral nucleicacid

Retrovirus

Figure 12.13

•12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

•How Restriction Fragments Reflect DNA Sequence

– Restriction fragment length polymorphisms (RFLPs)• Reflect differences in the sequences of DNA samples

Crime scene Suspect

w

x

y y

z

CutCut

Cut

DNA from chromosomes

CCGG

GGCC

ACGG

TGCC

CCGG

GGCC

CCGG

GGCC

Figure 12.11A

•12.9 DNA microarrays test for the expression of many genes at once

– DNA microarray assays• Can reveal patterns of gene expression in

different kinds of cells

CONNECTION

– DNA microarray

1 mRNAisolated

Reverse transcriptaseand fluorescent DNAnucleotides

2 cDNA madefrom mRNA

4 UnboundcDNA rinsedaway

3 cDNA appliedto wells

DNA microarrayEach well contains DNAfrom a particular gene

Actual size(6,400 genes)

Nonfluorescentspot

Fluorescentspot

cDNA

DNA of anexpressed gene

DNA of anunexpressed gene

Figure 12.9

•12.10 Gel electrophoresis sorts DNA molecules by size

+ +

– –

Powersource

Gel

Mixture of DNAmolecules ofdifferent sizes

Longermolecules

Shortermolecules

Completed gel

Figure 12.10

– After digestion by restriction enzymes• The fragments are run through a gel

–

+

Longerfragments

Shorterfragments

x

w

y

z

y

1 2

Figure 12.11B

•12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

•Using DNA Probes to Detect Harmful Alleles– Radioactive probes

• Can reveal DNA bands of interest on a gel

– Detecting a harmful allele using restriction fragment analysis

1

2

3

4

5

Restriction fragment preparation

Gel electrophoresis

Blotting

Radioactive probe

Detection of radioactivity(autoradiography)

I II III

I II III

Restriction fragments

Filter paper

Probe

Radioactive, single-stranded DNA (probe)

Film

IIIIII

I

II

IIIFigure 12.11C

DNA technology is used in courts of law

CONNECTION

•DNA and Crime Scene Investigations– Many violent crimes go unsolved

• For lack of enough evidence

– If biological fluids are left at a crime scene• DNA can be isolated from them

– DNA fingerprinting is a set of laboratory procedures• That determines with near certainty whether two

samples of DNA are from the same individual

• That has provided a powerful tool for crime scene investigators

Investigator at oneof the crime scenes(above), Narborough,England (left)

DNA Fingerprinting1st-The DNA molecule is cut with restriction enzymes

2nd- we have to separate the fragments

This is done by a technique called gel electrophoresis

The DNA is placed on a tray filled with gel through which an electric current runs causing the fragments to move through the gel. The segments separate by how far they move in the gel according to size.

The DNA will form bands corresponding to the bases (and no

two people have the same sequence of bases) in the gel which are unique for each individual. This is DNA fingerprinting

– DNA fingerprinting can help solve crimes

Defendant’sblood

Blood fromdefendant’s clothes Victim’s

blood

Figure 12.12A Figure 12.12B

Safety and ethical issues

• Methods for purifying the DNA

• Vectors for carrying the DNA into cells and replicating it

• Techniques for determining nucleotide sequences of DNA molecules