Chapter 20 Notes: DNA Technology

Understanding & Manipulating Genomes

• 1995: sequencing of the first complete genome (bacteria)

• 2003: sequencing of the Human Genome mostly completed

• These accomplishments depended on new technology:– Recombinant DNA: DNA from 2 sources (often 2 species)

are combined in vitro into the same DNA molecule• Called Genetic engineering: direct manipulation of genes for

practical purposes

DNA technology has launched a revolution in the area of:

BIOTECHNOLOGY: the use of living organisms or their components to do practical tasks

-microorganisms to make wine/cheese

-selective breeding of livestock

-production of antibiotics-agriculture

-criminal law

**Practical goal of biotech = improvement of human health and food production

Ch 20 looks at:

1. Main techniques for manipulating DNA

2. How genomes are analyzed & compared at the DNA level

3. Practical applications of DNA technology (including social & ethical issues)

“Toolkit” for DNA technology involves:

-DNA vectors

-host organisms

- restriction enzymes

VECTORS = carriers for moving DNA from test tubes back into cells

-bacterial plasmids (small, circular DNA molecules that replicate within bacterial cells)

-viruses

HOST ORGANISMS:bacteria are commonly used as hosts in genetic engineering because:

1) DNA can easily be isolated from & reintroduced into bacterial cells;

2) bacterial cultures grow quickly, rapidly replicating any foreign genes they carry.

RESTRICTION ENZYMES = enzymes that recognize and cut short, specific nucleotide sequences (called restriction sites)

-in nature, these enzymes protect the bacterial cell from other organisms by cutting up their foreign DNA

Restriction Enzymes (cont.)…most restriction sequences are

symmetrical in that the same sequence of 4-8 nucleotides is found on both strands, but run in opposite directions

restriction enzymes usually cut phosphodiester bonds of both strands in a staggered manner producing single stranded “sticky ends” 

Restriction Enzymes (cont.)…“sticky ends” of restriction

fragments are used in the lab to join DNA pieces from different sources (complementary base pairing)

*RECOMBINANT DNA

unions of different DNA sources can be made permanent by adding DNA ligase enzyme (form covalent bonds between bases)

DNA Technologies:

1) Cloning

2) DNA fingerprinting (profiling)

3) Microarray

4) Gene therapy

Steps Involved in Cloning a Human

Gene:1)  Isolate human gene to clone (ex: insulin);

2) Isolate plasmid from bacterial cell;

3) cut both DNA samples with the same restriction enzyme to open up bacterial plasmid & create sticky ends on both samples;

4) Mix the cut plasmids and human DNA genes & seal with DNA ligase;

plasmid

Human gene

Cloning a Human Gene (cont.)…5) Insert recombinant DNA plasmid back into

bacterial cell;

6) As bacterial cell reproduces, it makes copies of the desired gene;

-grow cells on a petri dish

7) Identify cell clones carrying the gene of interest.

-HOW? Which ones took up the gene & are making insulin?

*Add a 2nd gene besides insulin; add one for antibiotic resistance & then grow bacteria on that antibiotic

LE 20-4_3

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

Plate the bacteria on agarcontaining ampicillin and X-gal.Incubate until colonies grow.

Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carryingrecombinantplasmid withdisrupted lacZ gene

Bacterialclone

Why can bacteria produce insulin through recombinant DNA technology? The genetic code is universal!!!!