Download Biotechnology

Biotechnology
CHAPTER 20
You Must Know

The terminology of biotechnology.

How plasmids are used in bacterial transformation to clone genes.

The key ideas that make PCR possible and applications of this
technology.

How gel electrophoresis can be used to separate DNA fragments or
protein molecules.

Information that can be determined from DNA gel results, such as
fragments sizes and RFLP analysis.
DNA sequencing and cloning

DNA cloning yields multiple copies of a gene or other DNA
segment.

Biotechnology: is the process of manipulating organism or their
components for the purpose of making useful products.
 Including
earlier practices like selective breeding of farm animals and
using microorganisms to make cheese and wine.

Genetic engineering: is the process of manipulating genes
and genomes.

Recombinant DNA: is DNA that has been artificially made,
using DNA from different sources- often different species. An
example is the introduction of a human gene into an E. coli
bacterium.
DNA cloning and its application

Gene cloning: is the process by which scientists can produce multiple
copies of specific segments of DNA that they can then work with in the lab.

Many bacteria have DNA outside the main circular chromosome in
plasmids.

Plasmids: is a small, circular extra-chromosomal loop of DNA. Plasmids are
often used in biotechnology.

Plasmids only have a small number of genes, which are useful to the
bacterial is in a particular environment but may not be required for survival
or reproduction of the bacteria.

Gene cloning is useful for two basic purposes:

To make copies of, or amplify, a particular gene.

Produce a protein product.
Cloning Genes in
Bacterial Plasmids

1. Gene inserted into plasmid (cloning vector).

2. Plasmid put into bacterial cell.

3. Host cell grown in culture to form a clone of cells
containing the “cloned” gene of interest.

4. Basic research and various application.

Gene for pest resistance inserted into plants.

Gene used to alter bacteria for cleaning up toxic
waste.

Protein dissolves blood clots in heart attach therapy.

Human growth hormones treats stunted growth.

Restriction Enzymes: are used to cut out stands of DNA at
specific locations (called restriction sites).

They are mostly derived from bacteria where they serve the
important function of protection against invading viruses.

When a DNA molecule is cut by restriction enzymes, the result
will always b e a set of restriction fragments, which may have at
least one single-stranded end, called a sticky end.

Sticky ends can form hydrogen bonds with complementary
single-stranded pieces of DNA.

These unions can be sealed with the enzyme DNA ligase.
Using restriction Enzymes to
make Recombinant DNA

Gene cloning relies on restriction
enzymes to cut DNA molecules at a
limited number of specific locations.

Restriction enzymes recognizes a
particular short DNA sequence,
restriction site, cutting both DNA strands
at precise points within the restriction
site.
What can you do with cloned genes?

Nucleic acid hybridization- if the sequence of the gene is
known a probe can be made to track the gene.
 Probes-
short sequences of bases that are complimentary to part of
the gene and are radioactive or fluorescent.
 For
example, if the known sequence is G-G-C-T-A-A, then we would
synthesize the complementary probe C-C-G-A-T-T.
 Making
it radioactive we can trace it.
Probes

PCR- polymerase chain reaction)
amplifies a particular piece of DNA.

Allows us to do other techniques with
a small sample of DNA.
Gel electrophoresis

Is a lab technique used to separate
macromolecules, primarily DNA and
proteins.

The principles of this separation of
DNA include.

1. An electric current is applied to the
field. DNA is negatively charged and
migrates to the positive electrode.

2. A gel mad of a polymer is used as a
matrix to separate molecules by size. The
gel allows smaller molecules to move
more easily than larger fragments of
DNA.

The DNA must be stained or tagged for
visualization.

Restriction fragment length polymorphisms (RFLPs)- result from small
differences in DNA sequences and can be detected by
electrophoresis.

The differences in banding patterns after electrophoresis allows for
diagnosis of disease or is used to answer paternity and identity
questions.

This process leads to a genomic library.

Genomic library- is a set of thousands of recombinant plasmid clones,
each of which has a piece of the original genome being studied.

A cDNA library is made up of complementary DNA made from mRNA
transcribed by reverse transcriptase.

This technique rides the gene of introns but may not contain every
gene in the organism.

Genomic library- is a set of thousands
of recombinant plasmid clones, each
of which has a piece of the original
genome being studied.

A cDNA library is made up of
complementary DNA made from
mRNA transcribed by reverse
transcriptase.

This technique rides the gene of
introns but may not contain every
gene in the organism.
Study gene expression and function

Genome-wide studies of gene expression are made possible by the
use of DNA microarray assays.

1. Small amounts of ssDNA fragments representing different genes
are fixed to a glass slide in a tight grid, termed a DNA chip.

2. The mRNA molecules from the cells being tested are isolated and
used to make cDNA using reverse transcriptase, then tagged with a
fluorescent dye.

3. The cDNA bonds to the ssDNA on the chip, indicating which
genes are “on” in the cell (actively producing mRNA). This enables
researchers, for example, to see differences in gene expression
between breast cancer tumors and noncancerous breast tissue.
Basic research and other applications

In animal cloning the nucleus of an egg is removed and
replaced with the diploid nucleus of a body cell, a process
termed nuclear transplantation. The ability of a body cell to
successfully form a cone decreases with embryonic
development and cell differentiation.

The major goal of most animal cloning is reproduction, but
not for humans. In humans, the major goal is the production
of stem cells.

Stems cells can both reproduce itself indefinitely and
produce other specialized cells. Stem cells have enormous
potential for medical applications.
 Cloning

Embryonic stem cells are pluripotent, which means “capable of
differentiating into many different cell types”.

-multipotent stem cells – more differentiated than embryonic, but
still capable of becoming several different types of cells

It can be used to repair cells for people with diabetes or certain
types of brain cells for people with Parkinson’s disease.
Practical Applications

Diagnosis of disease: Use RFLP analysis
(cystic fibrosis, sickle-cell disease) or
through amplification of blood
samples to test for viruses (HIV).

Gene therapy: Alteration of an
afflicted individual’s genes. It holds
great potential for treating disorders
traceable to a single defective gene,
such as cystic fibrosis.

The production of pharmaceuticals:
Gene splicing and cloning can be
used to produce large amounts of
particular proteins in the lab.

Transgenic: animals are created when eggs are fertilized in vitro and then a
desired gene is cloned and inserted into the nucleus of the embryo. If
successful, the transgenic

Forensic: DNA samples taken from blood, skin cells or hair of alleged
criminal suspects can be compared to DNA collected from the crime
scene.

Environmental cleanup: Scientists engineer metabolic capabilities into
microorganisms, which are then used to treat environmental problems,
such as removing heavy metals from toxic mining sites.

Agricultural: GMO’s-certain genes that produce desirable traits have
been inserted into crop plants to increase their productivity or efficiency.