Chapter 14DNA Technologies

Recombinant DNA Recent technology – still changing New combinations of DNA – usually a DNA

sequence inserted into bacteria Allows DNA sequence to be copied and

amplified (a.k.a. cloned)

Cloned DNA Clone – a collection of molecules or cells, all

identical to an original molecule or cell To clone a gene – make many identical copies of it,

often by placing it in a culture of bacteria The cloned gene can be a normal copy (‘wild type’)

or an altered version (‘mutant’) Recombinant DNA technology makes gene cloning

possible The goals of gene cloning are: study of specific

genes and genetic engineering

Overview: Gene Cloning1. Restriction enzymes are used to cut DNA

molecules in specific places

2. The fragments of DNA are incorporated into a vector, such as a bacteriophage or plasmid, which can carry it into a host cell

3. The recombinant DNA is replicated and distributed to daughter cells during cell division

Step 1: Restriction enzymes Enzymes found in bacteria – are helpful to them to fight

foreign, invading DNA There are thousands of different restriction enzymes These enzymes cut DNA at specific sites These cleavage sites are usually at a 4 or 6 base-pair

palindromic sequence The ‘top’ strand from 5’ to 3’ is the same as the ‘bottom’

strand from 5’ to 3’ Cleavage that cuts the two strands directly across from each

other leaves blunt ends Cleavage that is uneven leaves single-stranded tails called

sticky ends

Restriction enzymes…Cuts usually occurs at

a palindromic sequence

SmaI: produces blunt ends

5´ CCCGGG 3´ 3´ GGGCCC 5´

EcoRI: produces sticky ends

5´ GAATTC 3´ 3´ CTTAAG 5´

Examples of Palindromes:Don't nod

Dogma: I am GodNever odd or even

Too bad – I hid a bootRats live on no evil starNo trace; not one carton

Was it Eliot's toilet I saw?Murder for a jar of red rum

Some men interpret nine memosCampus Motto: Bottoms up, Mac

Go deliver a dare, vile dog!Madam, in Eden I'm AdamOozy rat in a sanitary zooAh, Satan sees NatashaLisa Bonet ate no basil

Do geese see God?God saw I was dog

Dennis sinned

Restriction enzymes… The names are derived from the names of the bacteria they

originally came from They always cut DNA in the same place, regardless of the

source of the DNA Enzymes with staggered cuts complementary ends

(overhangs are ‘sticky’) These result in complementary ends that can be ligated

together Enzymes that cut at the same position on both strands leave

blunt ends DNA fragments with blunt ends can also be ligated together,

but with lower efficiency

Step 2: Incorporation into vectors Plasmids are cleaved by restriction enzymes

and have sticky or blunt ends Foreign DNA fragments (cut in the same

places so they have the same ends) can be linked to the ends with DNA ligase and inserted into the plasmids

This produces the recombinant DNA

Useful vectors…plasmids Plasmids are naturally occurring extra-chromosomal

DNA molecules found in bacteria They are circular and double-stranded They are the means by which antibiotic resistance is

often transferred from one bacteria to another (remember the mice in Griffith’s experiments?)

They do not usually contain genes essential to the bacteria under normal conditions

Useful vectors… viruses Bacteriophages are viruses that infect

bacteria These can also be made to carry recombinant

DNA into bacteria for cloning Engineered viruses can also be used as

vectors into cells of eukaryotic organisms

Step 3: Replication of Recombinant DNA The cells containing the recombinant DNA are

grown in culture – replicating the new DNA as they do

Not all the cells will be descendants of those with the recombinant DNA these need to be eliminated from the culture

For this reason, plasmids that also confer resistance to a particular antibiotic or allow cells to use a specific nutrient are often used

This allows scientists to separate transformed cells from untransformed cells

Why recombinant DNA? This technology has been used to:

Add favorable genes to agricultural crops, for example pest or herbicide resistance

Modify bacteria to produce enzymes used in industry, for example the enzyme used for making cheese

Produce therapeutic products such as human insulin and blood clotting factors

In the future we hope to: Provide gene therapy – the goal is to introduce the

correct version of a gene into the cells of a patient to correct protein production sickle-cell, cystic fibrosis, and some eye diseases

Silence overactive genes through RNA interference (RNAi) – uses a double-stranded RNA molecule to stop protein production of a targeted gene lupus, Crohn’s disease, autism, Alzheimer’s, rheumatoid

arthritis, Parkinson’s disease

Other DNA Technologies Polymerase Chain Reaction (PCR)

Uses nucleotides and primers, along with heat, to copy tiny amounts of DNA many times in vitro

Useful for analysis of crime scene evidence or other times when additional DNA is desired

Sensitive to contamination from outside DNA Gel electrophoresis

Allows us to separate DNA fragments based on their movement through a gel submitted to an electrical field

The smallest fragments of DNA migrate faster (and therefore further) from the starting point

This is the basis for ‘DNA fingerprinting’