DNA Technology• Genetic Engineering – direct manipulation of

genes for practical purposes–Scientists can make recombinant DNA and

then introduce it into cultured cells that replicate the DNA and may express its genes, yielding a desired protein.

–Often, E. coli is used as the “host”• Biotechnology – manipulations of organisms

to make useful products.• Recombinant DNA – genes from two different

sources are combined in vitro (outside the living body in a lab).

Techniques for Gene Cloning

Techniques for Gene Cloning• Plasmid – circular DNA that replicates within

bacterial cells (separate from bacterial chromosomes)

1. Isolation of plasmid

2. Gene insertion into plasmid

3. Plasmid put into bacterial cell

4. Cell cloned

5. Identification of desired clone

6. Copies of the gene/Copies of the protein• Used in gene therapy (HGH) and agriculture

(resistance to pests)

Restriction Enzymes• Restriction enzymes cut up foreign DNA –

recognize short nucleotide sequences• Restriction site – area recognized by

restriction enzyme (5’ – 3’)• Restriction fragments – nucleotide

sequence left after DNA has been cut.• Sticky end – single-stranded DNA fragment;

DNA ligase can permanently fuse fragments together

Restriction Enzymes

Transgenic organism

An organism that contains genes from other species.

Results from the formation of recombinant DNA.

Ex: Fruit fly w/firefly gene for luciferase

Bacterial Transformation•Another method used to form transgenic organisms•Plasmids inserted to change the proteins produced by cells•Lab 6A•pGLO

DNA Cloning

DNA Cloning• Cloning Vector – the original bacterial

plasmid• Procedure: Cloning a Eukaryotic Gene in

a Bacterial Plasmid• The E. coli plasmid has two useful genes:

ampR (resistant to ampicillian) and lacZ (catalyses hydrolysis of lactose)

1. Isolation of vector (bacterial plasmid) and gene-source DNA (human tissue)

DNA Cloning2. Insertion of DNA into the vector

• Restriction enzyme cuts plasmid DNA at single restriction site

• Cuts human DNA making thousands of fragments

• One of these fragments carries the gene we want

• The restriction enzyme creates compatible sticky ends on both the human DNA fragments and the plasmid DNA

• The DNA and plasmids are combined – DNA ligase “glues” the pieces together

DNA Cloning3. Introduction of cloning vector into cells

• Transformation – uptake of naked DNA from surrounding solution

• Some acquire the desired DNA; others take up other DNA

4. Cloning of cells• Put bacterial cells on a plate containing medium

with ampicillin and X-gal• Bacteria with recombinant plasmids carrying

foreign DNA with form white colonies on medium containing ampicillin and X-gal

DNA Cloning

5. Identification of cell colonies carrying the gene of interest

• Nucleic acid hybridization – base pairing between the gene and a complementary sequence using a nucleic acid probe

• The probe can be labeled with a radioactive isotope or a fluorescent tag

• Then the strand of DNA is separated by denaturing it with heat or chemicals

• The desired DNA can then be isolated an grown in large amounts

• A complete set of thousands of recombinant plasmid clones

• Can be stored as a plasmid library (bacterial cells) or phage libraries (viruses)

Cloned Genes Are Storedin DNA Libraries

• Any piece of DNA can be quickly copied many times without using a host cell

• DNA is placed in a test tube with:– Special DNA polymerases (first isolated from

bacteria growing in hot springs – the enzyme does not denature with heat)

– Supply of nucleotides– Synthetic single-stranded DNA that serves as

primers• Can be used to amplify a specific gene prior to

cloning

Polymerase Chain Reaction (PCR) Clones DNA In Vitro

• PCR can be used to quickly amplify DNA from a 40,000 year old woolly mammoth; tiny amounts of blood, tissue, or semen from a crime scene; and DNA of viral genes from HIV-infected cells

DNA Analysis and Genomics

• Suppose we have cloned a DNA segment carrying a human gene of interest – now we begin to ask some far-reaching questions:

1. Do genes differ in different people, and are certain alleles associated with a hereditary disorder?

2. Where and when is it expressed?

3. What is its location within the genome?

4. Evolutionary questions - Species to species differentiation?

DNA Analysis and Genomics• Genomics – the study of whole sets of genes

and their interactions• Gel Electrophoresis – method of sorting DNA

molecules into bands, each consisting of molecules of the same length– Separation on the basis of size, electrical charge,

and other physical properties• Restriction fragments – detect DNA

differences that affect restriction sites– Gel Electrophoresis used to sort DNA fragments

by size after being treated with restriction enzymes

Gel Electrophoresis

1. Digest two different DNA samples with same restriction enzyme (the samples should differ in one or more restriction site)

2. Use nucleic acid hybridization with a specific probe to label discrete bands that derive from our gene of interest (usually used in combination with Southern blotting)

Gel Electrophoresis

Gel Electrophoresis

Gel ElectrophoresisThe DNA fingerprints below represent four different individuals.

Which of the following statements is consistent with the results? • B is the child of A and C.• C is the child of A and B

Which of the following are probably siblings? • A and D • C and D

Entire Genomes Can Be Mapped at the DNA Level

• Human Genome Project – begun in 1990 to map all the human genes

• Surprisingly, there are few genes in the Human Genome

Practical Application of DNA Technology

• Diagnosis of Diseases– PCR can be used to amplify and detect HIV DNA

in blood or tissue samples– Scientists can diagnose hundreds of human

genetic disorders before the onset of symptoms, even before birth

– We can identify symptomless carriers of potentially harmful recessive alleles

– Genes have been cloned for many human diseases, including hemophilia, cystic fibrosis, and Duchenne muscular dystrophy

Practical Application of DNA Technology

• Human Gene Therapy – the alteration of an afflicted individual’s genes– Theoretically, it is possible to replace or

supplement the defective gene with a normal allele

– The new allele could be inserted into the somatic cells of the tissue affected by the disorder

– The cells that receive the normal allele must be ones that will continue to reproduce during the patient’s life – one example is stem cells from bone marrow that give rise to all blood and immune cells

Practical Application of DNA Technology

• Use of stem cells to promote healthy bone marrow

Practical Application of DNA Technology

• Pharmaceutical Products– Mostly proteins– Human insulin – Human growth hormone (HGH)– Tissue plasminogen activator (TPA) – protein that

helps dissolve blood clots (very expensive)– Drugs that mimic a receptor protein that HIV binds

to in entering white blood cells – the HIV binds to the drugs and fails to enter the blood cell

– Vaccines

• Reproductive Cloning– Producing complete, genetically identical

animals– Usually talked about in the press

• DOLLY the SHEEP.

Practical Application of DNA Technology

Practical Application of DNA Technology

• Forensic Uses – DNA fingerprinting

• Environment Uses– Sewage treatment plants rely on the ability of

microbes (bacteria and protists, mainly) to degrade many organic compounds into nontoxic forms

• Agricultural Uses– Animal Husbandry and “Pharm” Animals– Transgenic organisms – their genomes carry

genes from another specie

Practical Application of DNA Technology

• Agricultural Uses (continued)– Genetic engineering in plants (editable cotton

seed – 2006)– Delayed ripening– Resistance to spoilage and disease– Increase nutritional value (“golden rice”)

SO THE BIG QUESTION IS: