Biotechnology Chapter Four Lecture- DNA

Introduction toStudying 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

Learning Outcomes

The manipulation of genetic information,

DNA and RNA codes, is at the center of most biotechnology research and development.

Section 4.1- DNA Structure and Function

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.

Phosphodiester bonds

connect sugar molecules.

The phosphate groups have a

negative charge.

Nitrogenous bases are purines (two rings) or pyrimidines (single ring)

Nitrogenous bases from each strand bond to

each other in the center through H-bonds.

A with T (2 H-bonds) G with C (3 H-bonds)

Always purine with pyramidine giving the

double strand consistent width

The H-bonds are rather weak; therefore, the two strands of DNA

separate easily in high temperatures.

G/C rich DNA will separate at slightly higher temps due to

having more H-bonds

Similarities in DNA Molecules Among Organisms

All DNA molecules are composed of four nucleic monomersAdenosine deoxynucleotide (A), Cytosine deoxynucleotide (C),

Guanosine deoxynucleotide (G), and Thymine deoxynucleotide (T)

Virtually all DNA molecules form a double helix

The amount of adenosine equals the amount of thymineThe amount of guanosine equals the amount of cytosine

due to base pairing


DNA is antiparallel-

✦nucleotides in each strand are oriented in opposite direction of the other strand

✦one strand is 3’ to 5’ the other is 5’ to 3’4.


DNA is antiparallel-

✦nucleotides in each strand are oriented in opposite direction of the other strand

✦one strand is 3’ to 5’ the other is 5’ to 3’4.


10 base pairs / turn-The double helix has a regular shape which is recognized by other molecules.

4. 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 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 or two nitrogen-containing rings; forms the critical hydrogen bonds between opposing strands of a double helix

• Base pair – the 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

•

Vocabulary

• 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 involves 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

•

Vocabulary• 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

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

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

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

5. 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 DNAViruses are classified according to the type of cell they attack:

Bacterial (bacteriophages)Plant

Animal

4.2 Sources of DNA


Bacterial Cell CultureSome bacteria prefer a liquid medium (nutrient broth),

and some prefer a solid medium (nutrient agar).

4.2 Sources of DNA


Bacterial Cell CultureAgar plates contain nutrient broth gelled

with agar protein.

4.2 Sources of DNA


Bacterial Cell CultureAll media must be prepared under sterile

conditions.

4.2 Sources of DNA


Bacterial Cell Culture

Introduction of cells must be done under sterile conditions to ensure purity.

4.2 Sources of DNA



An autoclave sterilizes equipment and solutions with high pressure and

temp.

4.2 Sources of DNA



Sterile technique is an important lab skill for a microbiologist.

4.2 Sources of DNA



Sterile technique is doing something

without contamination by

unwanted organisms or their spores.

In prokaryotes the DNA is floating in the cytoplasm.

Bacterial genes

Bacterial genes

There is one large, twisted, circular chromosome that contains essential genes.

There may be plasmids- small circular DNA molecules with non-essential but often

advantageous genes

Bacterial genes

Plasmids are transferred between bacteria in nature and in the lab allowing easy transfer of

genes between prokaryotes.

Bacterial genes

Plasmids are good vectors for gene recombination. A vector is a vehicle used to transfer genetic material.

Bacterial genes

Prokaryotic DNA regulation is simple

Bacterial OperonAn operon contains the controlling elements that

turn genetic expression ON and OFF.


RNA polymerase attaches to the promoter.


Attachment of an inhibitor to the operator region will block production turning the

gene off.

Prokaryotic DNA regulation is simple-the lac operon in E.coli

Genes must be transferred along with appropriate regulatory sequences.

Eukaryotic DNA

Eukaryotes have multiple chromosomes

Eukaryotic DNAEach chromosome contains a single linear (very long) DNA molecule wrapped around

histone proteins.

Eukaryotic DNATightly coiled chromosomes hide genes.

Some unwinding from histones must occur for gene expression.

Eukaryotic DNA

There is much spacer DNA that does not code for anything.

Eukaryotic DNA

Eukaryotic Gene. Eukaryotic genes have a promoter to which RNA polymerase binds, but they do not have an operator region. Transcription factors may bind at

enhancer regions and increase gene expression.

Eukaryotic DNA

RNA polymerase moves down the DNA molecule until it reaches a structural gene which is

transcribed until a termination sequence is reached.

Eukaryotic DNA

The transcribed RNA molecule has introns (noncoding) removed and exons spliced together

which leave the nucleus as mRNA.

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 – the cells that have taken foreign DNA and started expressing the genes on the newly acquired DNA

•

Vocabulary

• 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

•

Vocabulary

• 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

• Agar – a 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

• Silencer – a section of DNA that decreases the expression of a gene

•

Vocabulary

• Transcription factors – molecules that work to either turn on or off the transcription eukaryotic genes

• 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

• Histones – the nuclear proteins that bind to chromosomal DNA and condense it into highly packed coils

•

Vocabulary

• Nonpathogenic – not known to cause disease

• Bacteriophages – the viruses that infect bacteria

• Gene therapy – the process of treating a disease or disorder by replacing a dysfunctional gene with a functional one

Vocabulary

4.2 Review Questions1. 1. Plasmids are very important pieces of DNA. How

do they differ from chromosomal DNA molecules?

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

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

4. 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)- What do you want to produce?

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

Basic steps of genetic engineering

4.3 Isolating and Manipulating DNA

3. Manipulation of the DNA instructions-change the DNA

or -move it to a new organism

4. Harvest the molecule or product and test it

To make a profit, the product must also be marketed.

Basic steps of genetic engineering

Recombinant DNA Technology

Recombinant DNA is made by combining different DNA molecules.

Recombinant DNA and the proteins produced from it have an “r” before their name.rhInsulin = recombinant human insulin




http://www.dnalc.org/view/15255-Producing-human-insulin-using-recombinant-DNA-Walter-Gilbert.html

http://www.youtube.com/watch?v=mhvARWPS1zM








































































































































































Site-Specific Mutagenesis

Process of inducing changes (mutagenesis) in certain sections (site-

specific) on a particular DNA code

Expose cells to chemicals, viruses, or radiation causing random mutations.Screen mutants for desirable traits.

Site-Specific MutagenesisRandom!

directed!!

Site-Specific Mutagenesis

http://www.youtube.com/watch?v=DjGLUnJSRYs

http://video.google.com/videoplay?docid=5044846172948251835




















































Process of correcting faulty DNA codes that cause genetic diseases

and disorders

Gene Therapy


and disorders

Manipulation must occur in mature multicellular organism

Gene Therapy


and disorders

Manipulation must occur in mature multicellular organism

Viruses are used to insert functional genes.

Gene Therapy

http://www.youtube.com/watch?v=gl2miunHTRI&feature=related





























































































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

(site-specific)

Vocabulary

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



isolation of the DNA sequence



isolation of the DNA sequence harvest of the molecule or product



isolation of the DNA sequence harvest of the molecule or product manipulation of the DNA instructions



isolation of the DNA sequence harvest of the molecule or product manipulation of the DNA instructions identification of the molecule to be produced



isolation of the DNA sequence harvest of the molecule or product 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?CFTR gene = cystic fibrosis transmembrane conductance regulator


4.4 Using Gel Electrophoresis to Study Gene Molecules


DNA is negatively charged and moves towards the + end of an electric field.



Moving molecules are separated by size in a “gel”. Smaller pieces travel farther.



Moving molecules are separated by size in a “gel”. Smaller pieces travel farther.

DNA is chopped up by restriction enzymes before loading in gel.


agarose gel- Medium to large DNA pieces (500bp - 25,000bp)

Poly-acrylamide (aka PAGE)- smaller stuff like protein, RNA, or small DNA

Components of Agarose Gel Electrophoresis

Powdered agarose + boiling TRIS buffer solution is made to specified concentration



0.8% is appropriate for most restriction enzyme digested DNA



0.8% is appropriate for most restriction enzyme digested DNA

3% agarose gels would be much slower running

A loading dye is used to visualize the movement of molecules.


A set of DNA fragments of known size are also run for comparison.


Gel Stains

Methylene blue makes the DNA visible.

Gel Stains

Ethidium bromide (EtBr)

makes DNA glow orange in UV

light.More sensitive

More hazardous

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.

Molecules in a Gel Box. For the gel box to conduct electricity, the solution in the gel box

must contain ions.Sodium chloride (NaCl) solution can be used,

but other salts, such as TRIS or lithium, dissolved in water (called a “running buffer”),

are better for conducting electricity.

• 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•

Vocabulary

• 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•

Vocabulary

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?




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?

Biotechnology Chapter Four Lecture- DNA

Education

strands of dna

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dna replicates

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