DNA

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Chapte

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Honor

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DNA Technology

Technology used to help with genetic engineering helps us:

1. identify genes for specific traits2. transfer genes for a specific trait from one organism to another

3. cure disease4. treat genetic disorders5. improve food crops

How can you get a desired trait without directly manipulating the organisms DNA?

1.HYBRIDIZATION; crossing organisms of different traits to produce a hardier product ex: mule

2.INBREEDING/SELECTIVE BREEDING; maintain the present genes by breeding only within the population ex: pedigree animals

3.INDUCING MUTATIONS; radiation, chemicals polyploidy (3N or 4N) plants resulted larger and hardier

Now let us manipulate the genes by altering the organisms DNA

a) DNA Technology: sci. involved in the ability to manipulate genes/DNA

a) Cure diseaseb) treat genetic disordersc) Improve crops

Tools:

1. DNA extraction

2. Restriction enzymes

3. Gel electrophoresis

4. DNA ligase

5. Polymerase chain rxn. (PCR)

Method: (5 steps)

1.Extract gene insulin2.Cut insulin producing gene out using “restriction

enzymes”1.Sticky ends create overhang2.Blunts no overhangs

3.Cutting clone vector cut plasmid with same restriction enzyme

4.Ligation: donor gene is spliced into plasmid DNA, DNA ligase glues it(this forms recombinant DNA = plasmid DNA + new piece of DNA)

5.Plasmid returned to bacterium & reproduces using donor gene in it (this is transgenic organism = organism with foreign DNA incorporated in it’s genome)

6.*reproduce*

Restriction Enzymes

BACTERIAL ENZYMES are used to cut DNA molecule into more manageable pieces

They recognize certain sequences

Creating “single-chain” tails in DNA called STICKY ENDS

Sticky Ends

Readily bind to complimentary chains of DNA therefore pieces of DNA that have been cut with the same restriction enzyme can bind together to form a new sequence of nucleotides

Recognizes CTTAAG

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Cloning Vectors

Cloning vector is a carrier that is used to clone a gene and transfer it from one organism to another.

Many bacteria contain a cloning vector called a PLASMID.

PLASMID is a ring of DNA found in a bacterium in addition to its main chromosome.

PROCEDURE

To be used as a cloning vector in gene transfer experiments a plasmid is isolated from a bacterium.

Using restriction enzymes the plasmid is then cut and a DONOR GENE (specific gene isolated from another organism is spliced into it)

Then the plasmid is returned to the bacterium, where it is replicated as the bacterium divides, making copies of the donor gene.

Plasmid now contains a GENE CLONE

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Cloning Vectors

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Plasmid

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Transplanting genes

In some cases, plasmids are used to transfer a gene to bacteria so that the bacteria will produce a specific protein

Ex: INSULIN = protein that controls sugar metabolism

Bacteria that receives the gene for insulin will produce insulin as long as the gene is not turned off

Steps:

1. ISOLATING A GENE – isolate the DNA from human cells and plasmids from the bacteria

Use restriction enzymeSplice human DNA into plasmids to create a genomic library (set of thousands of DNA pieces from a genome that have been inserted into a cloning vector)

Steps cont…

2. PRODUCING RECOMBINANT DNA = combination of DNA from 2 or more sources

Inserting a donor gene such as human gene for insulin, into a cloning vector, such as bacterial plasmid results in a recombinant DNA molecule!

Steps cont…

3. CLONING DNA – the plasmid containing recomb. DNA is inserted into a host bacterium (called transgenic organism

The trans. Bact. Is placed in a nutrient medium where it can grow and reproduce.

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Expression of Cloned Genes

Sometimes PROMOTERS must also be transferred so the genes will be turned on.

Genes are often turned off until the proteins they code for are needed.

Practical uses of DNA Technology

1. Pharmaceutical products: insulin, HBCF (human blood clotting factor)

2. Genetically engineered vaccines

3. Increased agriculteral yields

4. Improving quality of produce1.Slow down ripening2.Enhance color3.Reduce fuzz4.Increase flavor5.Frost resistance

Negatives

AllergiesLabel’s don’t include all

informationMay create “super weeds”

Gene therapy

Treatment of genetic disorders

Ex: cystic fibrosis

DNA Technology Techniques

I. DNA Fingerprints pattern of bands made up of specific fragments from an individual’s DNA

USED FOR:

DETECTION OF A RELATIVESIMILARITIES BETWEEN SPECIES

How do you make DNA fingerprints?RFLP (restriction fragment length

polymorphism) analysis1. extract DNA from specimen using restriction enzymes

2. separate fragments of DNA using electrophoresis (separates DNA according to size and charge)

3. placed in wells made on gel4. electric current run through gel

Continue…5. negative fragments migrate to positive charged end of gel but not all at same rate

6. pores in gel allow smaller fragments to migrate faster separating fragments by size.

7. blotted onto filter paper.

Can you tell if this could be the father?

Accuracy of DNA Fingerprints

DNA fingerprints are very accurate

However, genetic tests can only absolutely disprove, not prove, relationship!

Courts accept 99.5% accuracy as proof of alleged paternity

Polymerase Chain Reaction (PCR)

Used when you only have a TINY piece of DNA

PCR can be used to quickly make many copies of selected segments of the available DNA

Use a PRIMER to initiate replication

DNA doubles every 5 minutes

PCR is used for:1. crimes2. diagnosing genetic disorders from embryonic cells

3. studying ancient fragments of DNA (tiny amounts)

HUMAN GENOME PROJECT

2 GOALS:1. determine nucleotide

sequence of entire human genome (aprox 3 billion nucleotide pairs or about 100,000 genes

2. map the location of every gene on each chromosome

19961 % of 3 billion nucleotide

pairs of DNA human genomes were analyzed

This allows for us to identify and determine the function of 16,000 genes!

Gene TherapyTreating a genetic disorder by

introducing a gene into a cell or by correcting a gene defect in a cell’s genome.

Ex: Cyctic fibrosis cause one defective gene malfunction of one protein

Gene Therapy for Cyctic FibrosisNasal spray carrying normal

cyctic fibrosis gene to cells in nose and lungs

Must repeat treatment periodically

Ethical IssuesMany people worry about how personal genetic information will be

used:

Insurance???Employment????

Human Genome Project will undoubtedly involve ethical decisions about how society should use the information! WHAT DO YOU THINK??

Practical Uses of DNA Technology1. produce perscription drugsVaccine (harmless version of a virus or a bacterium)

Pathogen (disease causing agent) treated chemically or physically so that they can no longer cause disease.

Pathogen (Ag) Antibody (Ab)DNA tech. may produce vaccines safer than traditional ones!

Increasing Agricultural Yields

DNA Tech. used to develop new strains of plants

Ex: scientists can make tomato plants toxic to hornworms and effectively protect the plant from these pests.

See the hornworm beginning to form at the leaves!

This hornworm eats and destroys the tomato plant!

Hornworms attack tomato plants

Crops that do NOT need fertilizerPlants require NITROGEN to

make proteins and nucleic acidsMost plants get their N from the

soilTRANSGENIC FOOD CROPS

contain genes for nitrogen fixation so they can grow in nitrogen POOR soil.

Genetically Engineered Foods

Foods may have toxic proteins or substances causing ALLERGIES

Ex: changing the gene that codes for an enzyme to ripening in tomatoes they are able to make tomatoes ripen without becoming SOFT!!

Genetically Engineered Crops

Some are concerned that genetically engineered crops could spread into the wild and wipe out native plant species.

SUPERWEEDS!!!!!!!!