DNA TECHNOLOGYthe new genetics

Chapter 13

Ch 13 VOCABULARYput a + by the terms you know and – by the ones

you don’t. GMO Clone Vaccine biotechnolo

gyGenetic 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?

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.

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)

The Human Genome Project

Goals of the Human Genome Project:

• Determine the nucleotide sequence of the entire human genome.

• Map the location of every gene on each chromosome.

• Compare the genomes of other organisms to the human genome to understand:1. How genomes are organized.2. How gene expression is controlled.3. How cellular growth and

differentiation are under genetic control.

4. How evolution occurs.

GENOMIC LIBRARYis a catalog of the DNA of a species

• Cut up the DNA of the species into tiny pieces using restriction enzyme.

• Put each DNA fragment into a different cloning vector- ex. plasmid.

• Put each recombinant plasmid into a separate bacterium.

• FREEZE until needed.

Gene Therapy

• Treating a genetic disorder by introducing a gene into a cell or by correcting a gen defect in a cell’s genome.

• 1990’s.• Cystic Fibrosis, AIDS, Ovarian

Cancer.

Practical Uses of DNA Technology:

• 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.

DNA technology can be used to:

1. Cure diseases2. Treat genetic

disorders3. Improve food crops

Golden rice contains beta-carotene, which our bodies use to make vitamin A.

Figure 20.18 “Pharm” animals secrete spider silk in their milk

Could use thisTechnology to Make insulin or Human growth Hormone

Figure 20.16 One type of gene therapy procedure

transgenic organism

• Contains new DNA.• Ex. Bacterium with plasmid containing

insulin gene.

• Grow bacteria (beaker or petri dish)• Bacteria express (transcribe/translate)

the cloned gene to make insulin.• Insulin is extracted (purified) from the

medium.• Treatment for Diabetes.

Injecting DNA into an embryo… how you create a “cloned organism”

SCNT Somatic Cell Nuclear Transfer

Tools for manipulating genes

Restriction Enzymes (endonucleases)

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

• Provide protection for bacteria against viruses.

3 examples of restriction enzymes:EcoRI, BamHI, HindIII

Restriction Enzymes or Restriction Endonucleases

• Are bacterial enzymes that cut DNA molecules into smaller pieces

• They cut the DNA at a specific site, a known sequence of DNA .

for example: CTTAAG GAATTC• EcoRI cuts between the G and A, leaving two

open ends with single-chain “tails” called “sticky ends”

Ex. CTTAA and G G AATTC

• Sticky ends readily bind to complementary chains of DNA.

• Thus, pieces of DNA that have been cut with the same restriction enzyme can bind together to form a new sequence of nucleotides.

Restriction Enzymes

CLONING VECTORS• Restriction enzymes can be used to isolate a specific

gene of interest from a donor called the donor gene.• A plasmid is a ring of DNA found in a bacterium in

addition to its main chromosome.• Cut the plasmid with the same restriction enzyme as

the donor gene to “splice” it into the plasmid. • Insert this recombinant DNA plasmid into the

bacterium.• When the bacteria reproduces by binary fission the

recombinant plasmid does too, we call this cloning a gene.

• When a virus is used as the vector for gene transmission, this is called transduction.

Plasmids: small circular pieces of DNA.

Plasmids often contain genes for antibiotic resistance.

Conjugation- when bacteria exchange plasmids.

Transformation:

• bacteria can incorporate new DNA into their genome.

• They do it all the time naturally.

• Pick up plasmids from their environment.

Transplanting Genes

• Plasmids are used to transfer a gene to bacteria so the bacteria will produce a specific protein.

• Ex. Human insulin Human Growth Hormone

Just give the bacteria food and they will reproduce and produce your protein. You have a protein factory!!!! (clean up your protein, separate it and purify it)

1. Plasmid & Donor gene (cut by same RE)

2. Spliced w/ ligase3. Recombinant

plasmid inserted into bacteria (transformed)

4. Bacteria replicates & produces

5. Gene clone- exact copy of the gene.

Cloning A Gene

Recombinant Organisms

• Organisms that receive the recombinant plasmid.

• Ex. Glo fish, glowing cat, pharm animals, golden rice, roundup ready soybeans,

How to create a recombinant plasmid

1) Treat plasmid and donor gene with the same restriction endonuclease (they used EcoRI)

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

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

Phosphodiester bonds link sugar-phosphates of nucleotides… hydrogen bonds form/break spontaneously.

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

DNA Technology Techniques

PCRGel Electrophoresis

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

What you need:• DNA sample• Free nucleotides

– A heat resistant DNA polymerase

– Example: Taq polymerase• Primers: short segments(20-

30bases) of DNA complementary to the ends of the DNA being copied.

Polymerase Chain Reaction1) Denature the original strand

of DNA with heat.

1) Cool the mixture, allowing the primers to bind (anneal) to the DNA.

1) 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.

1) 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 than longer ones.

Figure 20.x1a Laboratory worker reviewing DNA band pattern

DNA TECHNOLOGY TECHNIQUES USED TO ANALYZE DNA sequences

DNA Fingerprint• Pattern of bands, arranged in colums,

made up of specific fragments from an individual’s DNA.

• Can be used to: 1. compare samples of blood or tissue left

at a crime scene;2. determine how closely related species

are;3. Paternity testing.

Making a DNA FingerprintRFLP analysis

• 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.• Smaller fragments migrate the furthest 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.

APPLICATIONS 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!

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

Figure 20.17 DNA fingerprints from a murder case