C H A P T E R 2 0

BIOTECHNOLOGY

DNA CLONING YIELDS MULTIPLE COPIES OF A GENE

OR OTHER DNA SEGMENT20.1

BIOTECHNOLOGY

• Biotechnology – the manipulation of organisms or their components to make useful products

• Biotechnology is used in all facets of life, and the advances being made in this field are only growing

DNA AND BIOTECHNOLOGY

• DNA is the molecule that holds the instructions for life• Scientists have learned how to manipulate it, and insert

new pieces of DNA into pre-existing DNA• Recombinant DNA – DNA molecules formed when

segments of DNA from two different sources are combined in vitro

DNA CLONING

• Gene Cloning – The production of multiple copies of a single gene

• Often to clone a gene, a bacteria is used• The plasmid of the bacteria is more

easily manipulated, and a selected portion of DNA (or a gene) is inserted into this plasmid

• The bacteria then reproduce asexually, producing identical copies of their DNA

• Each new cell now has the desired gene

• This can also be used to produce large quantities of a desired protein (not just the DNA)

CLONING VECTORS

• Plasmids are also known as cloning vectors• Cloning Vector – a DNA molecule that can carry

foreign DNA into a host cell and replicate there

EXAMPLES OF GENE CLONING

• Genes for pest resistance has been inserted into plants• Genes have been inserted into bacteria that allow them

to break down oil spills• HGH has been produced using gene cloning• Proteins that have dissolve blood clots are produced

using gene cloning

RESTRICTION ENZYMES

• Plasmid DNA is cut using restriction enzymes• Restriction Enzymes – an enzyme that cuts DNA at specific

nucleotide sequences• In bacterial cells, restriction enzymes protect bacteria by cutting up

DNA that is foreign to the cell so that it cannot infect the bacteria• Scientists have found restriction enzymes that will actually cut the

bacterial plasmid, rather than foreign DNA

RESTRICTION ENZYMES

• Restriction enzymes will cut up DNA in to many pieces because, by chance, the nucleotide sequence it is designed to cut will occur many times in a molecule

• The resulting pieces are called Restriction Fragments

• Effecting restriction enzymes will cut the sugar-phosphate backbone to have at least one single stranded end known as a sticky end

• These sticky ends can be ‘glued’ into DNA using DNA Ligase

CUTTING DNA

• The DNA plasmid is first isolated• It is then cut by using restriction enzymes

(Imagine below that this is an entire circular plasmid)

G

CTTAA

C T T A A

G

C T T A A G

CTTAAG

G

CTTAA

C T T A A

G

Sticky End

Sticky End

The sticky ends allow outside pieces of DNA to be attached and inserted into the DNA

TRANSFORMATION

• Transformation – the change of a bacteria cell due to the uptake in incorporation of foreign DNA

• Whenever a bacterial cell takes in new DNA it is said to have been ‘transformed’

TRANSFORMING BACTERIA

The gene for HGH is removed from a

human cell

Plasmid

The plasmid is cut at certain sequences in the DNA using the

same Restriction Enzyme used to cut the HGH gene from a human

cell

The plasmid will have sticky ends in addition to the HGH gene

The gene for HGH is then mixed with

the bacterial plasmid, and the

HGH is incorporated into

the plasmid

DNA Ligase covalently bond

the sugar phosphate backbone

The plasmid is then added to the

bacteria

The bacteria now has the gene for HGH, and has the ability to produce it

This shows the process of transformation in bacteria

MARKERS

• Often times, genes can be used as markers to make sure that the bacteria have undergone transformation:• FGP – Bacteria will glow under IR light if the gene was

incorporated into the plasmid• AMP – Bacteria will be immune to the anti-biotic

ampicillin

DNA LIBRARIES

• Genomic Library – a collection of DNA fragments that are stored and propagated in a population of cloning vectors

• This allows researchers to study the entire genome of an organism

PCR

• Polymerase Chain Reaction – A method used to amplify a piece of DNA quickly, many times, and without using cells

• Beneficial if DNA segment is short• Often used in crime scenes

PCR - PROCESS

1. DNA is incubated in a test tube with a special DNA Polymerase, nucleotides, and primers

2. DNA is heated to a certain temperature so that the DNA Ladder separates

3. It is then cooled, and the Polymerase and primers replicate the DNA

4. This cycle is repeated over and over, doubling the amount of DNA every cycle

The number of DNA

molecules doubles

every cycle

The DNA multiplies

exponentially

DNA TECHNOLOGY ALLOWS US TO STUDY THE

SEQUENCE, EXPRESSION, AND FUNCTION OF A GENE

20.2

GEL ELECTROPHORESIS

• Gel Electrophoresis – Process that separates nucleic acids or proteins on the basis of charge, size, or other physical properties

• Restriction enzymes are used to cut DNA up into different pieces

• RFLP’s – ‘Restriction Fragment Length Polymorphisms’

• Even in the same chromosomes of different organisms, there are differences that are seen in Restriction sites

Reservoirs

DNA Mixed with different restriction enzymes are put into reservoirs on a gel electrophoresis pad

An electric current is then put into the gel.

The current moves from the negative end to the positive end, and takes the different lengths of

DNA with it

Negative

The larger pieces of DNA don’t go as far,

and the smaller pieces move farther and faster away from the original

location

The bands that are formed can then be compared to other samples of DNA to

eventually determine the genes in the DNA

sequence

Negative

USES FOR GEL ELECTROPHORESIS

• Identify different alleles based on DNA sequence• Determine whose DNA was found at a crime

scene• Determine the father of a child

READING THE SEQUENCE

• Dyes are added to specific nucleotides and put into the test tubes

• The dyes terminate DNA replication• Using gel electrophoresis, the different lengths of

DNA are separated

TT

TT G

TT CG

TT CG A

Dye Molecule

GENOMICS

• Study of entire genomes and how genes interact• Made possible through sequencing

DNA MICROARRAY ASSAYS

• Measure expression of thousands of genes at once

• Compare gene expression of different cell types, stages of development, species, etc.

PROTEOMICS

• Study of proteins made from genomes

• Genetic complexity is determined by proteins produced not number of genes• Ex: Apples have 57,386 genes;

humans have 20,000 genes• Apples produce 65,000

proteins; humans produce 100,000

CLONING ORGANISMS MAY LEAD TO PRODUCTION OF

STEM CELLS FOR RESEARCH AND OTHER APPLICATIONS

20.3

CLONING

• Gene cloning (recombinant DNA)

• Reproductive• Generate an organism that

has the same DNA as a current or previously existing animal

• Mice, pigs, sheep, rabbits

• Therapeutic• Embryo cloning for studying

human development, stem cells

SAFETY & ETHICAL QUESTIONS

• Just because we can does it mean we should?

THE PRACTICAL APPLICATIONS OF DNA

TECHNOLOGY AFFECT OUR LIVES IN MANY WAYS

20.4

MEDICAL APPLICATIONS

• Diagnosis of disease• PCR, primers detect specific pathogens or genetic

mutations that cause disease

• Gene therapy• Insert normal alleles into someone whose cells contain

defective alleles

PHARMACEUTICAL APPLICATIONS

• Insulin, HGH manufactured by bacteria containing human gene

• Pharmacogenomics• How your genes influence your body’s response to

medications• Designer pharmaceuticals?

FORENSICS

• Uses combination of techniques to prove guilt/innocence

• Small samples can be used for PCR, electrophoresis, Southern blotting, RFLP analysis

ENVIRONMENTAL CLEANUP

• Bacteria can be modified to break down heavy metals, oil, and other toxins

AGRICULTURAL APPLICATIONS

• Animals• Manipulated to produce

human proteins such as hormones, clotting factors

• Manipulate to produce more of what we need, such as milk or eggs

• Plants• Introduce pest resistance• Improve nutritional value

(“golden” rice)