Biotechnology• The manipulation of organisms or their genes

for – Basic biological research– Medical diagnostics– Medical treatment (gene therapy)– Pharmaceutical production– Forensics– Environmental clean up– Agricultural applications– Genetic components to make useful products

• Humans have been manipulating the genetics of organisms for thousands of years

Animal husbandry/breeding

Biotechnology today

• Genetic engineering - the direct manipulation of genes for practical purposes–Manipulation of DNA is often called

“Recombinant DNA”–Nucleotide sequences from two different sources are combined in vitro into the same DNA molecule

• Recombinant DNA techniques usually start with DNA Cloning– Scientists clone pieces of DNA (or entire

genes) in order to work with them in the laboratory

– There are multiple methods to clone pieces of DNA or genes

DNA Cloning using bacteria and plasmids

• One common method to clone DNA involves using bacteria (often E. coli)

• Many bacteria contain an extachromosomal piece of DNA called a plasmid– Separate from main chromosome– Can replicate independently– Occur naturally in bacteria– Can be passed between bacteria

DNA Cloning using bacteria and plasmids

• Plasmids can be genetically engineered by inserting gene from another cell (plasmid is now recombinant DNA)

• Plasmids are used as cloning vectors – a piece of DNA used to carry foreign DNA into a host cell

• Plasmid is inserted back into bacteria by Transformation

• Bacteria is allowed to reproduce producing many identical bacteria with the plasmid (and desired gene)

Fig. 20-2

DNA of chromosome

Cell containing geneof interest

Gene inserted intoplasmid

Plasmid put intobacterial cell

RecombinantDNA (plasmid)

Recombinantbacterium

Bacterialchromosome

Bacterium

Gene ofinterest

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Plasmid

Gene ofInterest

Protein expressedby gene of interest

Basic research andvarious applications

Copies of gene Protein harvested

Basicresearchon gene

Basicresearchon protein

Gene for pest resistance inserted into plants

Gene used to alter bacteria for cleaning up toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

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Transformation• Transformation was first performed in the

laboratory by Griffith and later by Avery, MacLeod, and McCarty

• Bacteria can take up DNA only during the period a the end of logarithmic growth – cells are said to be competent (can accept DNA that is introduced from another source)

• E. coli are frequently used for transformation

• E. coli competence can be induced under carefully controlled chemical growth conditions

• Plasmids can transfer genes and act as carriers for introducing DNA from other bacteria or from eukaryotic cells

• E. coli cell membrane is weakened using ice cold CaCL2

• E. coli cells are then “heat shocked” to induce them to take up the plasmid

• Sterile technique must be used• Transformation Lab – we will transform bacteria by

introducing a plasmid that will convey resistance to the antibiotic, ampicillin

• Ampicillin kills bacteria by interfering with their ability to make cell walls

Biologists use Enzymes to “Cut and Paste” DNA

• Two important enzymes that biologists use for genetic engineering are restriction enzymes and DNA Ligase– restriction enzymes cut DNA molecules at

specific DNA sequences– DNA Ligase stick lengths of DNA together– genetic engineers are able to use both enzymes to

“cut and paste” DNA• This is how plasmids are genetically

engineered

Using Restriction Enzymes to Make Recombinant DNA

• Restriction enzymes come from bacteria• In nature, bacteria use restriction enzymes

for protection to cut foreign DNA (from invading viruses)

• Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites

• A restriction enzyme usually makes many cuts, yielding restriction fragments

• The most useful restriction enzymes cleave the DNA in a staggered manner to produce sticky ends

• Sticky ends can bond with complementary sticky ends of other fragments

• DNA ligase can close the sugar-phosphate backbones of DNA strands

Figure 13.23-3

Restriction enzyme cutsthe sugar-phosphatebackbones.

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DNA3

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DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.

DNA ligaseseals the strands.

Sticky end

One possible combination

Recombinant DNA molecule

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GCA A TT

GGCCA

TTA

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GGCCA

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Restriction site

GGCCA

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DNA Fingerprinting

• Restriction Enzymes are also used for DNA fingerprinting (profiling)– Creating a pattern of DNA bands on a gel

• Because the restriction site (recognition sequence) usually occurs (by chance) many times on a long DNA molecule, a restriction enzyme will make many cuts

• Result: production of fragments of DNA of various lengths – Restriction Fragment Length Polymorphs (RFLPs)

• Since all individuals have unique sequences of DNA, restriction enzymes cut each individual’s DNA into different sized RFLPs

• The RFLPs are then separated by gel electrophoresis resulting in a bar-like pattern

• Electrophoresis means “to carry with an electric current”

• Different sized RFLPs will be carried different distances by an electric current as they migrate through an agarose gel inside a gel box – Electricity is run through the gel box creating a

positive end and a negative end• Negatively charged DNA migrates from the

negative end of the gel box through the pores in the gel to the positive end of the gel box

• Smaller RFLPs will migrate farther than larger pieces, spreading the RFLPs across the gel in a bar-like pattern

• Stain is used to make the DNA bands visible

Figure 13.24Mixture ofDNA mol-ecules ofdifferentsizes

Cathode

Restriction fragments

Anode

Wells

Gel

Powersource

(a) Negatively charged DNA molecules will movetoward the positive electrode.

(b) Shorter molecules are impeded less thanlonger ones, so they move faster through the gel.

SEM photo of a 1% LE Agarose gel at 22kX magnification

Measuring fragment size• Compare bands to known

“standard”– Usually lambda phage cut

with HindIII• Nice range of size with a

distinct pattern

DNA Fingerprinting – Uses • Also called DNA Profiling• Used to reveal a DNA pattern which is unique

to an individual• Crimework: rape and murder cases (forensics)• Paternity suits• Missing persons and unidentified bodies• Immigration disputes• Animal work - breeding

Polymerase Chain Reaction• cloning a gene through genetic engineering can be time-

consuming and requires an adequate DNA sample as starting material

• PCR technique allows researchers to amplify a tiny sample of DNA millions of times in a few hours

• DNA polymerase uses nucleotides and primers to replicate a DNA sequence in vitro, thereby producing two molecules

• Two strands of each molecule are then separated by heating and replicated again, so then there are four, double-stranded molecules

• After the next cycle of heating and replication there are eight molecules, and so on

• Number of molecules doubles with each cycle• PCR is useful in amplifying tiny samples of DNA ranging

from crime scenes to archaeological remains

Figure 13.25

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Cycle 1yields 2 molecules

Genomic DNA

Denaturation

Target sequence

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Primers

New nucleotides

Annealing

Extension

Cycle 2yields 4 molecules

Cycle 3yields 8 molecules;

2 molecules(in white boxes)

match target sequence

Technique

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