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14 Δεκ 2012 (πριν από 4 χρόνια και 7 μήνες)

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Introduction to

Studying DNA


Chapter 4

Learning Outcomes


Describe the structure and function of DNA and explain the process by
which it encodes for proteins


Differentiate between eukaryotic and prokaryotic chromosomal
structure and explain how this difference impacts gene regulation in
the two cell types


Differentiate between bacterial cultures grown in liquid and solid media
and explain how to prepare each media type using sterile technique


Discuss the characteristics of viruses and their importance in genetic
engineering


Explain the fundamental process of genetic engineering and give
examples of the following applications: recombinant DNA technology,
site
-
specific mutagenesis, and gene therapy


Describe the process of gel electrophoresis and explain how the
characteristics of molecules affect their migration through a gel

4.1 DNA Structure and Function

The manipulation of genetic information, DNA
and RNA codes, is at the center of most
biotechnology research and development.

The Central Dogma of Biology.
Proteins are produced when genes on
a DNA molecule are transcribed into mRNA, and mRNA is translated into
the protein code. This is called “gene expression.” At any given moment,
only a relatively small amount of DNA in a cell is being expressed.

DNA Structure.
The
nucleotides in one chain of
the helix face one direction,
while those in the other
strand face the other
direction. Each nucleotide
contains a sugar molecule,
a phosphate group, and a
nitrogenous base.
Nitrogenous bases from
each strand bond to each
other in the center through
H
-
bonds. The H
-
bonds are
rather weak; therefore, the
two strands of DNA
separate easily in high
temperatures.

Similarities in DNA Molecules Among Organisms

1.
All DNA molecules are composed of four nucleic monomers

1.
Adenosine deoxynucleotide (A)

2.
Cytosine deoxynucleotide (C)

3.
Guanosine deoxynucleotide (G)

4.
Thymine deoxynucleotide (T)

2.
Virtually all DNA molecules form a double helix

3.
The amount of adenosine equals the amount of thymine


The amount of guanosine equals the amount of cytosine

4.
Nucleotides in each strand are oriented in the opposite direction of the
other strand

5.
Nitrogenous bases

6.
DNA undergoes semiconservative replication

DNA Replication.
DNA replicates in a
semiconservative
fashion in which one
strand unzips and
each side is copied. It
is considered
semiconservative since
one copy of each
parent strand is
conserved in the next
generation of DNA
molecules.

Variations in DNA Molecules


The number of DNA strands in the cells of an organism


The length in the base pairs of the DNA strands


The number and type of genes and noncoding regions


The shape of the DNA strands

Vocabulary


Chromatin



nuclear DNA and proteins


Gene


a section of DNA on a chromosome that contains the genetic code of a protein


Nitrogenous base


an important component of nucleic acids (DNA and RNA), composed of
one of two nitrogen
-
containing rings; forms the critical hydrogen bonds between opposing
strands of a double helix


Base pair



two nitrogenous bases that are connected by a hydrogen bond; for example, an
adenosine bonded to a thymine or a gaunine bonded to a cytosine


Phosphodiester bond



a bond that is responsible for polymerization of nucleic acids by
linking sugars and phosphates of adjacent nucleotides


Hydrogen bond


a type of weak bond that involved the “sandwiching” of a hydrogen atom
between two fluorine, nitrogen, or oxygen atoms; especially important in the structure of
nucleic acids and proteins


Pyrimidine


a nitrogenous base composed of a single carbon ring; a component of DNA
nucleotides


Purine


a nitrogenous base composed of a double carbon ring; a component of DNA
nucleotides


Antiparallel


a reference to the observation that strands on DNA double helix have their
nucleotides oriented in the opposite direction to one another


Semiconservative replication


a form of replication in which each original strand of DNA
acts as a template, or model, for building a new side; in this model one of each new copy goes
into a newly forming daughter cell during cell division

4.1 Review Questions

1.
Describe the relationship between genes, mRNA, and proteins.

2.
Name the four nitrogen
-
containing bases found in DNA molecules and
identify how they create a base pair.

3.
The strands on a DNA molecule are said to be “antiparallel.” What does
antiparallel mean?>

4.
During cell division, DNA molecules are replicated in a semiconservative
manner. What happens to the original DNA molecule during
semiconservative replication?

4.2 Sources of DNA

In nature, DNA is made in cells.

Mammalian Cell Culture


Growing mammalian cells in culture is more challenging than
growing bacterial cells


Mammalian cells are grown in a broth culture

Viral DNA

Viruses are classified according to the type of cell they attack:


Bacterial (bacteriophages)


Plant


Animal

Prokaryotic DNA

Bacterial Operon.

An operon contains the controlling
elements that turn genetic expression ON and OFF.

Bacterial Cell Culture

Eukaryotic DNA

Eukaryotic Gene.

Eukaryotic genes have a promoter to which
RNA polymerase binds, but they do not have an operator region.

Vocabulary


Medium


a suspension or gel that provides the nutrients (salts, sugars, growth factors,
etc.) and the environment needed for cells to survive; plural is media


Lysis


the breakdown or rupture of cells


R plasmid


a type of plasmid that contains a gene for antibiotic resistance


Transformed


refers to those cells that have taken foreign DNA and started expressing the
genes on the newly acquired DNA


Vector


a piece of DNA that carries one or more genes into a cell, usually circular as in
plasmid vectors


Operon


a section of prokaryotic DNA consisting of one or more genes and their controlling
elements


RNA polymerase


an enzyme that catalyzes the synthesis of complementary RNA strands
from a given DNA strand


Promoter


the region at the beginning of a gene where RNA polymerase binds; the
promoter “promotes” the recruitment of RNA polymerase and other factors required for
transcription


Operator


a region on an operon that can either turn on or off expression of a set of genes
depending on the binding of a regulatory molecule


Beta
-
galactosidase


an enzyme that catalyzes the conversion of lactose into
monosaccharides

Vocabulary


Agar


solid media used for growing bacteria, fungi, plant, or other cells


Media preparation


the process of combining and sterilizing ingredients (salts, sugars,
growth factors, pH indicators, etc.) of a particular medium


Autoclave


an instrument that creates high temperature and high pressure to sterilize
equipment and media


Enhancer


a section of DNA that increases the expression of a gene


Intron


the region on a gene that is transcribed into an mRNA molecule but not expressed in
a protein


Exon


the region of a gene that directly codes for a protein; it is the region of the gene that
is expressed


Transcription factors


molecules that work to either turn on or off the transcription
eukaryotic genes


Histones


nuclear proteins that bind to chromosomal DNA and condense it into highly
packed coils


Nonpathogenic


not known to cause disease


Bacteriophages


viruses that infect bacteria


Gene therapy


the process of treating a disease or disorder by replacing a dysfunctional
gene with a functional one

4.2 Review Questions

1.
Plasmids are very important pieces of DNA. How do they differ
from chromosomal DNA molecules?

2.
Bacteria cell DNA is divided into operons. Describe an operon
using the terms promoter, operator, and structural gene.

3.
Describe the human genome by discussing the number and types
of chromosomes, genes, and nucleotides.

4.
What is gene therapy? Cite an examples of how it can be used.

4.3 Isolating and Manipulating DNA

1.
Identification of the molecule(s)

2.
Isolation of the instructions (DNA sequence/genes) for the
production of the molecule(s)

3.
Manipulation of the DNA instructions

4.
Harvesting of the molecule or product, testing it, and marketing it

Recombinant DNA Technology

Methods to create new DNA molecules

Site
-
Specific Mutagenesis

Process of including changes (mutagenesis) in certain sections (site
-
specific) on a
particular DNA code

Gene Therapy

Process of correcting faulty DNA codes that cause genetic diseases and
disorders

Vocabulary


Bioremediation


the use of bacteria or other organisms to restore
environmental conditions


Site
-
specific mutagenesis


a technique that involves changing the genetic
code of an organism (mutagenesis) in certain sections (site
-
specific)

4.3 Review Questions

1.
Genetic engineering by any method requires certain steps. Put
the following steps in the correct order:


isolation of the instructions (DNA sequence/genes)


harvest of the molecule or product; then marketing


manipulation of the DNA instructions


identification of the molecule to be produced

2.
What “naming” designation is used with recombinant products
made through genetic engineering?

3.
What is the smallest change in a DNA molecule that can occur
after site
-
specific mutagenesis? What effect can this change
have?

4.
What gene has been the target of CF gene therapy? What does
this gene normally do?

4.4 Using Gel Electrophoresis to Study Gene Molecules

Components of Gel Electrophoresis


Powdered agarose


Boiling buffer solution

Agarose Gel Concentrates

Most commonly used when separating pieces of DNA
no smaller than 50 bp and no larger than 25,000 bp

Gel Stains

The gel is “run” until molecules of different sizes
are thought to have completely separated.

Agarose Gel Tray.

Gel trays differ depending on
the manufacturer. Each has some method of sealing
the ends so that liquid agarose can mold into a gel.
Some gel trays, such as those made by Owl
Separation Systems, make a seal with the box, so
casting a gel is simple. Other trays require masking
tape on the ends to make a mold. Still others, like
the one shown here, have gates that screw into
position: up for pouring the gel and down for running
the gel.


Molecules in a Gel Box.
If negatively
charged molecules are loaded into the
wells and run on the gel, the smaller
ones run faster and farther than the
larger ones toward the positive
electrode. This is because smaller
molecules pass more easily through the
tiny spaces of the gel network.

Vocabulary


Gel electrophoresis


a process that uses electricity to separate charged molecules,
such as DNA fragments, RNA, and proteins, on a gel slab


Agarose


a carbohydrate from seaweed that is widely used as a medium for
horizontal gel electrophoresis


Polyacrylamide


a polymer used as a gel material in vertical electrophoresis; used
to separate smaller molecules, like proteins and very small pieces of DNA and RNA


Ethidium bromide


a DNA stain (indicator); glows orange when it is mixed with
DNA and exposed to UV light; abbreviated EtBr


Methylene blue



a staining dye/indicator that interacts with nucleic acid molecules
and proteins, turning them to a very dark blue color


High through
-
put screening


the process of examining hundreds or thousands of
samples for a particular activity

4.4 Review Questions

1.
Agarose gels can be used to study what size of DNA fragments?

2.
If agarose gel material is labeled 1%, what does the 1% refer to?

3.
What causes molecules to be separated on an agarose gel?

4.
Name two common DNA stains that are used to visualize DNA on
agarose gels.

Questions and Comments?