BIOTECHNOLOGY UNIT:• “The Immortal Life of Henrietta Lacks”• Dr. Wayne Grody- Guest Speaker• “GATTACA”• Ch18&19 Pro&Eukaryotic control of gene expression• Ch11 Cell Signaling Cell Communication• Ch20&21 Biotechnology…manipulating microorganisms to do our

bidding!!!! Tools/techniques• LAB 6 MOLECULAR BIOLOGY

– Bacterial transformation– Restriction Enzymes/DNA Digests– Gel Electrophoresis (DNA fingerprinting)

• BIOTECH RESEARCH PAPER & SYMPOSIUM

Control of Gene Expression

Chapter 18

VOCABULARY

Hetero-chromatin

Eu-chromatin

Methylation Transcrition factors

Histonesnucleosomes

Operon/Operator/Promoter

Gene experssion

Inducible/Repressible

ATPADPAMP cAMP

Jacob & Manod

Galactosidase

Glucose Galactose Lactose Transcription factor

Negative regulation

Positive regulation

Inducer/Corepressor

CRP Regulatory Gene

VOCABULARY

QUESTIONS TO ANSWER

Who? What? Where? When? Which? Why? How?

Q & A• Q:What is gene expression?• A: Activating a gene to produce a protein.• Q: What is an operon?• A: The genes for creating an enzyme and the genes that

regulate their transcription.• Q: What makes up an operon?• Answer:

1) Promoter region2) Operator region3) Structural genes

OPERONPromoter, Operator, Structural Genes

Control of gene expression

• The expression of genetic material controls cell products, and these products determine the metabolism and nature of the cell.

• Gene expression is regulated by both environmental signals and developmental cascades or stages.

• Cell signaling mechanisms can also modulate and control gene expression.

• Thus, structure and function in biology involve two interacting aspects: the presence of necessary genetic information and the correct and timely expression of this information.

Gene expression can be under:

a) Negative regulation: When the operon is turned off by chemicals.

repressible or inducible or

b) Positive regulation: When gene expression is stimulated by chemicals.

2 Types of Negative Regulation Part 1: The trp operon

(repressible operon)• What does repressible mean?

• This operon is located in E. coli bacteria.

• The purpose of the operon is to create the enzymes that synthesize the amino acid tryptophan.

• When the operon is “ON:

1) RNA polymerase binds to the promoter region.2) RNA polymerase crosses over the operator region because the repressor is inactive and therefore does not bind to the operator.3) The structural genes are transcribed and tryptophan is synthesized.OPERON “ON”

Switching the trp operon “off”• Done by a repressor: a protein that binds to the operator region. • It blocks the attachment of RNA polymerase to the promoter region.1) A gene called a regulatory gene (located away from the operon)

produces the trp operon repressor.2) The repressor protein is allosteric having an active and inactive

state/shape.

3) At first the repressor protein is in its inactive form.4) When the amino acid tryptophan binds to the repressor

the repressor becomes its active form and binds to the operator region… blocking transcription.

5) Since tryptophan assists in turning the operon off it is called a co-repressor.

6) When the levels of tryptophan drop the repressor loses its tryptophan, changes shape, and the repressor is released from the operator , initiating transcription again.

TURNING THE OPERON OFF

regulatory gene operator

Regulatory gene codes for the repressor which may bind to the operator

Types of Negative Regulation Part 2: The lac operon(inducible operon)

• What does inducible mean?• This operon is also located in E. coli bacteria.• It was first discovered by: Francois Jacob and Jacques Monod (1961)

called the “Jacob and Monod model”• The purpose of the operon is to produce the enzyme

B-galactosidase that splits (via hydrolysis) lactose into glucose and galactose.

• This operon is normally off because the repressor protein is formed in its active shape, thus binds to the operator region blocking RNA polymerase.

• If the RNA polymerase is being blocked how does transcription ever occur???

Done by an inducer: a molecule that binds to and inactivates the repressor.1) The molecule allolactose (an isomer of lactose) binds to the

repressor, inducing an allosteric change.

2) The repressor is released from the operator region.

3) The RNA polymerase can move along the template strand catalyzing the synthesis of mRNA.

4) When the lactose levels decrease the repressor binds to the operator region and transcription is shut down.

Figure 18.21a The lac operon: regulated synthesis of inducible enzymes

Figure 18.21b The lac operon: regulated synthesis of inducible enzymes

Compare and contrast:

• The enzymes produced by the trp operon are called repressible enzymes and are involved in anabolic pathways.

• The enzymes produced by the lac operon are called inducible enzymes and are involved in catabolic pathways.

Types of Positive Regulation: A closer look at the lac operon

• In order for the lac operon to produce enzymes in large quantities, a second factor must exist…

• a low concentration of glucose.

How does E. coli sense the low levels of glucose and how is this relayed to the lac operon?

By the molecule cyclic AMP or cAMP.cAMP is present in large quantities when the glucose levels are low.1) The cAMP binds to an allosteric protein called cAMP receptor protein or

CRP2) The activated CRP binds to a site within the lac promoter adjacent to the

TATA box.3) The attachment of CRP makes it easier for RNA polymerase to bind to the

promoter region.

Figure 18.22a Positive control: cAMP receptor protein

Figure 18.22b Positive control: cAMP receptor protein

CRP is known as an activator protein because it activates transcription.

If the levels of glucose increase the levels of cAMP decrease and the CRP is released from its binding site.

Figure 18-22x cAMP

Figure 18.20b The trp operon: regulated synthesis of repressible enzymes (Layer 2)

Figure 18.21b The lac operon: regulated synthesis of inducible enzymes

Figure 18.22a Positive control: cAMP receptor protein

Figure 18.22b Positive control: cAMP receptor protein

The Organization and Control of Eukaryotic Genomes

Chapter 19

How is eukaryotic gene expression different from prokaryotic gene expression?

1. Importance of cell specialization in multicelluar Euk’s.2. Greater size of genome of Euk’s and chromatin structure:-single, circular, chromosome in PROKARYOTES-double, linear, protein enhanced in EUKARYOTES*

Histones/Nucleosomes = DNA is coiled around bundles of 8 or 9 histone proteins to form DNA-histone complexes called nucleosomes.

1. Euchromatin = regions where DNA is loosely bound to nucleosomes and is actively transcribed.

2. Heterochromatin = regions where nucleosomes are more tightly compacted and DNA is inactive. (stains darker)

* Compactly organized as chromosomes during cell division.

Figure 19.1 Levels of chromatin packing

DNA Packing is the first level of control of eukaryotic gene expression

Figure 19.0 Chromatin in a developing salamander ovum

Only _3_% of eukaryotic DNA is translated into

protein products, compared to almost

_100_% of prokaryotic DNA.

Sizewise…several million nucleotide

pairsvs.

2 x 10 8 pairs per chromosome

(that’s x 46 in humans!)

Figure 19.x1a Chromatin

HETEROCHROMATIN EUCHROMATIN

• Repetitive DNA = noncoding segments not transcribed within a gene

» CENTROMERE- center» TELOMERE- ends (telomerase)» PSEUDOGENES = not transcribed, almost identical to

a coding gene. May represent evolutionary precursor—mutated over the years.

• TRANSPOSONS or “jumping genes”= can move to a new location on the same chromosome or to a different chromosome. Discovered by Barbara McKlintock (maise)

Have the effect of a mutation… can change the expression of a gene

1. Turn on or off its expression2. Have no effect at all

Enduring understanding 3.B: Expression of genetic information involves cellular

and molecular mechanisms.

• Essential knowledge 3.B.1:

• Gene regulation results in differential gene expression, leading to cell specialization.

Enduring understanding 3.B: Expression of genetic information involves cellular

and molecular mechanisms.

• Essential knowledge 3.B.1:

• Gene regulation results in differential gene expression, leading to cell specialization.

Both DNA regulatory sequences, regulatory genes, and small regulatory RNAs are involved in gene expression.

• Regulatory sequences are stretches of DNA that interact with regulatory proteins to control transcription. (ex. promoter, terminator, enhancer)

• A regulatory gene is a sequence of DNA encoding a regulatory protein (repressor & activator) or RNA (miRNA & siRNA) blocks translation on a transcribed mRNA by binding to it.– Micro RNA– Small Interfering RNA

– RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules.

– Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from producing a protein. RNA interference has an important role in defending cells against parasitic nucleotide sequences – viruses and transposons – but also in directing development as well as gene expression in general.

In eukaryotes, gene expression is complex and control involves regulatory genes, regulatory elements and transcription

factors that act in concert.• Transcription factors bind to specific DNA

sequences and/or other regulatory proteins.• Some of these transcription factors are

activators (increase expression), while others are repressors (decrease expression).

• The combination of transcription factors binding to the regulatory regions at any one time determines how much, if any, of the gene product will be produced.

Controlling Eukaryotic Gene Expression

• Under positive control• Transcription will not take

place without the assembly of the transcription complex

• Transcription Factors are regulatory proteins that bond to the enhancer region, the promoter (TATA box) and to each other.

• Once the transcription factors have assembled around the promoter, they are called a transcription complex.

Areas that regulate eukaryotic transcription:

a) The Enhancer Region: causes the chromosome to loop and make contact with the Promoter regions.

• Located thousands of nucleotides away from the promoter. • Activator proteins bind to the enhancer regions and then

to the transcription complex after the DNA loops. • When the activator proteins (special transcription factors)

bind to the transcription complex, RNA polymerase is positioned over the promoter region and the rate of transcription increases.

b) The Silencer Region: a repressor region.

• Located close to the enhancer region.

• Repressor Proteins bind to the silencer sites prevent the activator proteins from binding to the enhancer region.

Turning On A Gene

Turning On A Eukaryotic Gene

Figure 19.8 A eukaryotic gene and its transcript

ALTERNATIVE RNA SPLICING

Control of Translation

Protein Processing

cancer

• What is cancer?

• Unregulated cell growth and division.

• What causes cancer?

• Damage to the genes regulating the cell division cycle.

• Usually by carcinogens (cancer causing agents).

• Tumor = a cluster of cancerous cells

• Metastases = When cells leave the tumor, spread, grow new tumors.

• Sarcomas = Tumors of the cells in connective tissue, muscle or bone.

• Carcinoma = Tumors of cells in epithelial tissue like skin.

• The three deadliest human cancers:• Lung …smoking• Colorectal …diet• Breast…causes is still unknown, however

some forms are inherited as the genes BRCA1 and BRCA 2.

• Genes that cause cancer are called oncogenes.

• The normal versions of these genes are called proto-oncogenes and they code for proteins that stimulate normal cell growth and division.

• How do proto-oncogenes become oncogenes?• Translocation, or movement of fragments of

chromosomes(break off and attach somewhere else) that result in being around an active promoter.

• Amplification, or increasing the number of copies of the gene in the cell.

• Point mutations, change the sequence, creating mutant proteins

• Two genes that are significant: ras gene and the p53 gene.

• The ras gene creates a protein that influences the cell cycle.

• The mutated ras protein is hyperactive, leading to excessive cell division.

• The p53 gene becomes active when DNA is damaged and creates a tumor suppressing protein.

• Mutating the p53 gene can lead to the formation of tumors.

• Chemicals in cigarette smoke induce p53 mutations.

• 15% of the cancers worldwide are associated with viral infections. Certain viruses can insert oncogenes others may insert DNA into proto-oncogenes, turning them into oncogenes.

Figure 19.13 Genetic changes that can turn proto-ocogenes into oncogenes

Figure 19.14 Signaling pathways that regulate cell growth (Layer 1)

Figure 19.14 Signaling pathways that regulate cell growth (Layer 2)

Figure 19.14 Signaling pathways that regulate cell growth (Layer 3)

Figure 19.15 A multi-step model for the development of colorectal cancer

• The basic structure of viruses includes a protein capsid that surrounds and protects the genetic information (genome) that can be either DNA or RNA.

• Viruses have a mechanism of replication that is dependent on the host metabolic machinery to produce necessary viral components and viral genetic material.

• Some classes of viruses use RNA without a DNA intermediate; however, retroviruses, such as HIV, use a DNA intermediate for replication of their genetic material.

• Some viruses introduce variation into the host genetic material. – When the host is bacterial, it is referred to as lysogenesis; – whereas in eukaryotic cells, this is referred to as

transformation.

• Since viruses use the host metabolic pathways, they experience the same potential as the host for genetic variation that results from DNA metabolism.

SUMMARYViral & Bacterial Control of

Gene Expression

CHECK FOR UNDERSTANDING

Both positive and negative control mechanisms regulate gene expression

in bacteria and viruses. 1. The expression of specific genes can be turned on by the

presence of an _________. 2. The expression of specific genes can be inhibited by the

presence of a _________. 3. Inducers and repressors are small molecules that interact

with ____________ and/or regulatory sequences.4. Regulatory proteins inhibit gene expression by binding to

_________and blocking transcription (negative control).5. Regulatory proteins stimulate gene expression by binding

to DNA and stimulating transcription (___________) or binding to _________ to inactivate repressor function.

6. Certain genes are continuously expressed; that is, they are always turned “on,” e.g., the _________ genes.

Both positive and negative control mechanisms regulate gene expression

in bacteria and viruses. • The expression of specific genes can be turned on by the

presence of an inducer. • The expression of specific genes can be inhibited by the

presence of a repressor. • Inducers and repressors are small molecules that interact

with regulatory proteins and/or regulatory sequences.• Regulatory proteins inhibit gene expression by binding to

DNA and blocking transcription (negative control).• Regulatory proteins stimulate gene expression by binding to

DNA and stimulating transcription (positive control) or binding to repressors to inactivate repressor function.

• Certain genes are continuously expressed; that is, they are always turned “on,” e.g., the ribosomal genes.

Gene regulation accounts for some of the phenotypic differences between organisms

with similar genes. QUESTIONS:• describe the connection between the regulation of gene

expression and observed differences between different kinds of organisms.

• describe the connection between the regulation of gene expression and observed differences between individuals in a population.

• explain how the regulation of gene expression is essential for the processes and structures that support efficient cell function.

• use representations to describe how gene regulation influences cell products and function.

Gene regulation accounts for some of the phenotypic differences between organisms

with similar genes. QUESTIONS:1. describe the connection between the regulation of gene expression and observed differences

between different kinds of organisms. Structure and function in biology result from the presence of genetic information and the correct expression of this information.

2. describe the connection between the regulation of gene expression and observed differences between individuals in a population. The expression of the genetic material controls cell products, and these products determine the metabolism and nature of the cell. Most cells within an organism contain the same set of genetic instructions, but the differential expression of specific genes determines the specialization of cells.

3. explain how the regulation of gene expression is essential for the processes and structures that support efficient cell function. Some genes are continually expressed, while the expression of most is regulated; regulation allows more efficient energy utilization, resulting in increased metabolic fitness.

4. use representations to describe how gene regulation influences cell products and function. Gene expression is controlled by environmental signals and developmental cascades that involve both regulatory and structural genes. A variety of different gene regulatory systems are found in nature. Two of the best studied are the inducible and the repressible regulatory systems (i.e., operons) in bacteria, and several regulatory pathways that are conserved across phyla use a combination of positive and negative regulatory motifs. In eukaryotes, gene regulation and expression are more complex and involve many factors, including a suite of regulatory molecules.

DNA TECHNOLOGY AND GENOMICS

Chapter 20

Ch 20 & 21 VOCABULARYput a + by the terms you know and – by the ones you don’t.

GMO Clone Vaccine biotechnology Genetic engineering

GFP ligation plasmid endonuclease technology

PCR insulin Gene expression

Transgenic Gel electrophoresis

DNA Gene therapy Restriction enzyme

Somatic cellNuclear transfer

DNA Fingerprint

HGH Dolly vector Biomedical agriculture

RFLP interleukin ethical interferon transformation

GENERATE YOUR OWN QUESTIONS:1pt question words: Who? What? Where? When? 2pt question words: Which? How? 3pt question words: Why?

Genetic Engineering is the application of molecular genetics for practical purposes.

Uses:1) Identify genes for specific traits2) Transfer genes for a specific trait from one

organism to another.Tools for manipulating genes:1) Restriction enzymes (endonucleases)2) Cloning vector (bacterial plasmid)

Transgenic/Recombinant organisms contain DNA that was not part of their original genome.

Green fluorescent protein (GFP) is responsible for the green bioluminescence of the jellyfish Aequorea victoria.

This is a GM mouse!

5. The genetic composition of cells can be altered by incorporation of exogenous DNA into the cells. As a

basis for understanding this concept:

a.Students know the general structures and functions of DNA, RNA, and protein.

b. Students know how to apply base-pairing rules to explain precise copying of DNA during semiconservative replication and transcription of information from DNA into mRNA.

5. The genetic composition of cells can be altered by incorporation of exogenous DNA into the cells. As a

basis for understanding this concept:

c. Students know how genetic engineering (biotechnology) is used to produce novel biomedical and agricultural products.

d.* Students know how basic DNA technology (restriction digestion by endonucleases, gel electrophoresis, ligation, and transformation) is used to construct recombinant DNA molecules.

e.* Students know how exogenous DNA can be inserted into bacterial cells to alter their genetic makeup and support expression of new protein products.

Recombinant organisms contain DNA that was not part of their original

genome.

The green fluorescent protein (GFP) created by these transgenic mice is responsible for the green bioluminescence of the jellyfish Aequorea victoria.

This GMO is a GM mouse!

Practical Uses of DNA Technology:

• Gene Therapy

• Pharmaceuticals- HGH, Interferons, Interleukins etc.

• Vaccines- solution that contains a harmless version of a virus or bacterium to stimulate an immune response & formation of “memory” cells.

• Increased Agricultural Yields- ex. crops that don’t need fertilizer.

Ethical Issues

• Describe two potential safety and environmental problems that could result from genetic engineering.

Golden rice contains beta-carotene, which our bodies use to make vitamin A… normal rice does not. Vitamin A deficiency results in blindness & lowered immunity.

Figure 20.x2 Injecting DNA

SomaticCellNuclearTransfer

What does that mean?

“Pharm” animals create other species proteins secreted in their milk, ex. spider’s silk.

Figure 20.16 One type of gene therapy procedure

We owe the field of Genetic Engineering to the bacterial cell.

1) Plasmids: small circular pieces of DNA.

Plasmids often contain genes for antibiotic resistance, thus

protection from fungi.

Ex. Penicillin

Ampicillin, Amoxycillin

2) Conjugation:

• Bacteria exchanging plasmids.

• Pili = cytoplasmic extensions used to contact other bacteria.

• Plasmids are exchanged through the pili.

3) Restriction endonucleases

• Molecular “scissors” that cut DNA at specific sequences.

• Provide protection for bacteria against viruses.

* More details to follow.

4)Transformation:• bacteria can incorporate new

DNA into their genome from the external environment.

We will do this in the next lab

• Give E. Coli a plasmid that contains:

1) Gene for Ampicillin resistance protein

2) Gene for B galactosidase enzyme to digest xgal sugar to make blue protein.

How restriction endonucleases work

• The enzyme will recognize a specific sequence of DNA & cut it at that specific place in a specific way.

• For example E. coli bacteria have a restriction endonuclease called EcoRI.• It will recognize the site: 5’-- GAATTC--3’

3’--CTTAAG--5’• Many recognition sites are palindrome sequences- read the same forwards/backwards- see 2 strands.• It will cut the DNA between: G AATTC

CTTAA G• The results are “sticky ends” or short, single strands of bases.• If two complementary sticky ends pair up, they can be joined by DNA ligase.

Restriction Enzymes

RECOMBINANT PLASMIDS

• Stanley Cohen and Herbert Boyer created the first recombinant plasmids in 1973.

• In one of the first recombinant experiments with animal DNA, Cohen and Boyer spliced an amphibian gene into a bacterial plasmid.

1972UCSF & Stanford scientists met at a conference in Hawaii on bacterial plasmids. Over lunch of hot pastramisandwiches they decided to pool their resources. Within fourthe joint labs succeeded in cloning predeterminedsegments of DNA. This paved the way for a new, huge, International industry that has created products such as:Human growth hormome(HGH), human insulin, and a heart medication to remove blood clots.

How to create a recombinant plasmid

1) Treat plasmid and amphibian DNA with the same restriction endonuclease (they used EcoRI)

2) This creates the same sticky ends on plasmid and amphibian DNA.

3) Place both together with DNA ligase to join the amphibian gene with the plasmid.

The recombinant plasmid is inserted (transformed) into bacterial cells and the bacteria made amphibian mRNA.

But not the protein… we’ll see why in a minute.

You can clone the gene using the bacterial cells

When scientists create plasmids one problem they encounter is that

prokaryotes cannot modify mRNA by removing the eukaryotic intron

sequences.

Introns often prevent translation.

To overcome this problem, a DNA gene is created using the modified mRNA as a

template.

1. Modified mRNA is isolated from the cytoplasm.2. An enzyme called reverse transcriptase creates a strand of DNA

from the mRNA template.3. The newly synthesized strand of DNA can act as a template for the

complementary strand.4. This type of DNA, synthesized without the intron sequence is called

complementary DNA or cDNA.5. The cDNA can be successfully inserted into plasmids, transcribed

and translated by bacteria.

Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene

Cloning A Gene

Screening for the Recombinant Plasmid(How to find the few bacteria transformed w/ recombinant plasmid from the rest!!!!)

• A recombinant plasmid should contain:

• 1) A gene for antibiotic resistance.

• 2) A functional gene containing a restriction site.

– Ex. THE GFP gene

• Why?

You can do this two ways:

1. They are grown on a media containing an antibiotic.Only the bacteria that took up the plasmid will survive. 2. Knock out a gene- this let’s you know if you successfully spliced

your “gene of interest” into the plasmid in the first place.If a gene on the plasmid contains a restriction site, then that gene

will be rendered useless when the foreign gene of interest is inserted.

Ex. The organisms that don’t “Glow” have the recombinant plasmid and do… ex. make HGH.

For Example:• The "Z gene" on a plasmid produces an enzyme that

metabolizes the sugar X-Gal.• When the gene is functioning X-Gal is broken down into

a blue product. • If the restriction site is within the "Z gene" and the gene

of interest is inserted there, the bacteria that have this plasmid cannot metabolize X-gal.

• When they are cultured on a petri dish, using an X-gal media, these bacteria will appear white.

• Other bacteria that have a plasmid without the gene of interest will appear blue.

Other techniques:

• X Antibody staining

• X Radioactive DNA probe

• Restriction Digest of DNA/Gel Electrophoresis

DNA FINGERPRINT/ RFLP analysisRestriction Digest of DNA Restriction Fragments separated by Gel Electrophoresis

DNA FINGERPRINT Cut DNA at specific sequences w/ restriction enzymes.

Each different sample is cut at different locations- makes different sized fragmentsLoaded onto gel. Electric current runs through. Pulls - DNA to + charge.

Small fragments move fastest, Large fragments left at the top.Unique banding pattern forms

Making a DNA Fingerprint• A DNA sample is extracted from

nucleated cells.• The DNA is amplified using

P.C.R.• The DNA is cut into fragments by

restriction enzymes.• The stained fragments are placed

into a gel, and are moved by an electrical current.

• Comparison is made between DNA samples.

Paternity Testing: Who’s the daddy? A or B

Making a DNA Fingerprint… cont.

• Smaller fragments migrate the farthest and the result is a column of dark DNA bands that extend across the gel.

• The amount of DNA between restriction sites varies from individual to individual of the same species. The differences are called restriction fragment length polymorphisms or RFLP’s. RFLP’s result in unique restriction fragment patterns on a gel.

Using the circle provided, construct a labled diagram of the restriction map of the plasmid. Explain how you developed your map.

b) Describe how: recombinant DNA technology could be used to insert a gene of interest into a bacterium.

Recombinant bacteria could be identified. Expression of the gene of interest could be ensured.

b) Describe how: recombinant DNA technology could be used to insert a gene of interest into a bacterium. Recombinant bacteria could be identified. Expression of the

gene of interest could be ensured.

c) Discuss how a specific genetically modified organism might provide a benefit for humans and at the same time, pose a threat to a

population or ecosystem.

c) Discuss how a specific genetically modified organism might provide a benefit for humans and at the same time, pose a threat to a

population or ecosystem.

Polymerase Chain Reaction a way to make millions of copies of DNA!!!

What you need:1. DNA sample

2. Free nucleotides– A heat resistant DNA polymerase – Example: Taq polymerase

3. Primers: short segments(20-30bases) of DNA complementary to the ends of the DNA being copied.

What to do:1) Denature the original strand of

DNA with heat.2) Cool the mixture, allowing the

primers to bind (anneal) to the DNA.

3) The DNA polymerase binds free nucleotides to the primer using the original DNA strand as a template. This creates two copies of the DNA sample.

4) Repeat.

Gel Electrophoresis• Technique used to separate restriction

fragments.

• DNA fragments of different lengths are separated as they diffuse through a gelatinous material under the influence of an electric field.

• Since DNA is negatively charged (phosphate groups), it moves toward the positive electrode.

• Shorter fragments move further/faster than longer ones so a pattern is made.

Figure 20.15 RFLP markers close to a gene

Figure 20.x1a Laboratory worker reviewing DNA band pattern

Figure 20.x1b DNA study in CDC laboratory

APPLICATIONS of

Gel Electrophoresis

1. Compare DNA fragments of closely related species to determine evolutionary relationships.

2. CSI. Compare restriction fragments between individuals of the same species- murder, rape.

Fragments differ in length because of polymorphisms, slight differences in DNA sequences. These fragments are called restriction fragment length polymorphisms, or RFLP’s.

Figure 20.6 Genomic libraries

Figure 20.13 Alternative strategies for sequencing an entire genome

Table 20.1 Genome Sizes and Numbers of Genes

Figure 20.14a DNA microarray assay for gene expression

Figure 20.14b DNA microarray assay for gene expression

Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants

DNA, and in some cases RNA, is the primary source of heritable information.

CHECKING FOR UNDERSTANDING:

• Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. Q: What are three genetic engineering techniques?

• Illustrative examples of products of genetic engineering include: Q: What are three examples of products of Genetic

Engineering?

DNA, and in some cases RNA, is the primary source of heritable information.

• Genetic engineering techniques can manipulate the heritable information of DNA and, in special cases, RNA. – ex.Electrophoresis , Plasmid-based transformation ,

Restriction enzyme analysis of DNA , Polymerase Chain Reaction (PCR)

• Illustrative examples of products of genetic engineering include: – Genetically modified foods, Transgenic animals, Cloned

animals, Pharmaceuticals, such as human insulin or factor X