Chapter 12

Biotechnology manipulation of organisms or their

components to make useful products Wine, cheese Livestock selective breeding

DNA technology techniques used to study and manipulate

genetic material

manipulating genes for practical purposes Gene cloning

leads to the production of multiple, identical copies of a gene-carrying piece of DNA

Recombinant DNA formed by joining nucleotide sequences from two

different sources One source contains the gene that will be cloned Another source is a gene carrier

vector Plasmids often used

Restriction enzymes cut DNA at specific sequences Each enzyme binds to DNA at a different

restriction site Many restriction enzymes make staggered cuts

that produce restriction fragments with single-stranded ends “sticky ends”

Fragments with complementary sticky ends can associate with each other, forming recombinant DNA

DNA ligase joins DNA fragments together

A restrictionenzyme cutsthe DNA intofragments.

Restriction enzymerecognition sequence

Restrictionenzyme

Gene ofinterest

A DNA fragmentfrom anothersource is added.

Two (or more)fragments sticktogether bybase pairing.

StickyendSticky

end

DNA ligaseDNA ligasepastes thestrands together.

RecombinantDNA molecule

DNA1

2

4

5

3

1. Plasmid DNA isolated

2. DNA containing the gene of interest is isolated

3. Plasmid DNA is treated with a restriction enzyme

4. DNA with the target gene is treated with the same enzyme and many fragments are produced

5. Plasmid and target DNA are mixed and associate with each other

6. Recombinant DNA molecules produced when DNA ligase joins plasmid and target segments together

7. Recombinant plasmid containing the target gene taken up by a bacterial cell• Then binary fission!!

E. coli bacterium

Bacterialchromosome

A plasmidis isolated.

Gene ofinterest

The plasmid is cutwith an enzyme.

Plasmid

The cell’s DNAis isolated.

The cell’s DNA is cutwith the same enzyme.

DNA

Examples of gene use

A cell with DNAcontaining the geneof interest

Geneof interest

The targeted fragmentand plasmid DNAare combined.

DNA ligase is added,which joins the twoDNA molecules.

Geneof interest

Genes may be insertedinto other organisms.

The recombinant plasmidis taken up by a bacteriumthrough transformation.

Examples of protein use

Harvestedproteinsmay beuseddirectly.

The bacteriumreproduces.

Cloneof cells

Recombinantbacterium

RecombinantDNAplasmid

1

3

5

4

2

6

7

9

8

used to manufacture many useful products, chiefly proteins

Bacteria often used because: have plasmids and phages available for use as

gene-cloning vectors can be grown rapidly and cheaply can be engineered to produce large amounts of a

particular protein often secrete the proteins directly into their growth

medium

human insulin gene isolated and cut from its location on the human chromosome ◦ using a restriction enzyme

plasmid is cut using the same restriction enzyme

desired DNA (insulin gene) and plasmid DNA can be joined using DNA ligase

plasmid now contains the genetic instructions on how to produce the protein insulin

Bacteria can be artificially induced to take up the recombinant DNA plasmids and be transformed◦ successfully transformed bacteria will

contain the desired insulin gene transformed bacteria containing the insulin

gene can be isolated and grown As transformed bacteria grow they will

produce the insulin proteins coded for the recombinant DNA◦ Insulin harvested and used to treat diabetes

contain one or more genes introduced by artificial means

GM plants are being produced that are more resistant to herbicides and pests

provide nutrients that help address malnutrition

GM animals are being produced with improved nutritional or other qualities

Are they safe?!?!

DNA PROFILING

analysis of DNA fragments to determine whether they come from the same individual

compares genetic markers from noncoding regions that show variation between individuals

involves amplifying (copying) of markers for analysis

DNA isisolated.

1

2

3

The DNA ofselectedmarkers isamplified.

The amplifiedDNA iscompared.

Crime sceneSuspect 1 Suspect 2

Polymerase chain reaction (PCR)

method of amplifying a specific segment of a DNA molecule

PCR three-step cycle doubles the amount of

DNA in each turn of the cycle

Advantages:◦ amplify DNA from a small sample

◦ obtaining results rapidly

◦ highly sensitive, copying only the target sequence

used to separate DNA molecules based on size1. A DNA sample is placed at one end of a gel2. Current is applied and DNA molecules move

from the negative electrode toward the positive electrode

3. Shorter DNA fragments move through the gel matrix more quickly and travel farther through the gel

4. DNA fragments appear as bands, visualized through staining

5. Each band is a collection of DNA molecules of the same length

A mixture of DNAfragments ofdifferent sizes

Powersource

Gel

Completedgel

Longer(slower)molecules

Shorter(faster)molecules

In October 2001, Florida man died from inhalation anthrax◦ By the end of the year, four other people had also

died from anthrax Investigators analyzed the genome of the

anthrax spores used in each attack◦ Able to establish that the spores from all of the

cases were identical Suggested a single perpetrator of the crime Able to match the anthrax with one laboratory

subtype The Ames strain

Betty Anne Waters◦ Ayer, MA

1982 – brother arrested for murder Waters went to CCRI

◦ GED◦ Associates◦ Went to Roger Williams to get Bachelors and

Law degree Became brothers lawyer

◦ Witnesses lied****◦ DNA evidence in 1990’s◦ Innocence Project

Released in 2001 after serving 18 years in prison◦ $3.4 million dollar settlement

http://www.imdb.com/video/imdb/vi4273341977/

GENOMICS

study of an organism’s complete set of genes and their interactions

Initial studies focused on prokaryotic genomes Many eukaryotic genomes have since been

investigated

allows another way to examine evolutionary relationships showed a 96% similarity in DNA sequences

between chimpanzees and humans Functions of human disease-causing genes have

been determined by comparing human genes to similar genes in yeast

determining the nucleotide sequence of all DNA in the human genome

identifying the location and sequence of every human gene

revealed that most of the human genome does not consist of genes

Results indicate that: humans have about 20,000 genes in 3.2 billion

nucleotide pairs only 1.5% of the DNA codes for proteins, tRNAs,

or rRNAs remaining 98.5% of the DNA is noncoding DNA

Telomeres stretches of noncoding DNA at the ends of chromosomes

transposable elements, DNA segments that can move or be copied from one

location to another within or between chromosomes

Proteomics study of the full protein sets encoded by

genomes investigates protein functions and interactions

human proteome includes about 100,000 proteins

Human and chimp genomes differ by: 1.2% in single-base substitutions 2.7% in insertions and deletions of larger DNA

sequences

Genes showing rapid evolution in humans include genes for defense against malaria and

tuberculosis a gene regulating brain size the FOXP2 gene involved with speech and

vocalization