Genetic Engineering and Biotechnology

Genetic engineering – the direct manipulation of genes for practical purposes. ✄ Ex: “Improving” plants and animals by adding genes from other species. ✄ Rice with new genes for vitamins from bean. ✄ Cows with extra copies

of genes for milk. ✄ Glow-in-the-dark fish

(using genes from jellyfish) for the

home aquarium. ✄ Pink-haired poodles

for the pet market.

Definition

PCR is a technique by which any piece of DNA, however tiny, can be quickly amplified (copied many times) without using living cells. ✄ Amplify 40,000–year-old wooly mammoth DNA*. ✄ Amplify tiny amounts of DNA at a crime scene. ✄ Amplify embryonic DNA for pre-natal testing. ✄ Amplify DNA from difficult-to-detect viruses

like HIV.

Polymerase chain reaction

*

PCR consists of a series of 20 to 40 repeated temper- ature changes called cycles; each cycle doubles the amount of DNA. ✄ Step 1: Denaturation: heat to 94-98°C for 30 sec.

It breaks the hydrogen bonds.

Polymerase chain reaction

PCR steps (cont’d). ✄ Step 2: Extension/elongation:

DNA polymerase synthesizes a new DNA strand comple- mentary to the DNA tem- plate strand by adding nucleotides that are comple-

mentary to the template.

Polymerase chain reaction

PCR steps (cont’d). ✄ The procedure

repeats for 20 40 cycles, making making billions of

copies automatically.

Polymerase chain reaction

PCR machine

To learn the characteristics of DNA and protein samples, the material must first be separated. ✄ In gel electrophoresis, fragments of DNA or pro- teins move in an electric field and are separated according to their size.

Gel electrophoresis

Vertical separation

Horizontalseparation

Separation of DNA using an electric current.

Gel electrophoresis

Small-sized pieces move faster through the gel.

Gel electrophoresis of DNA is used in DNA profiling ✄ Large portions of any single person's DNA are the same as every other person's since they code for species-specific traits such as feet, ears, etc. ✄ Other fragments are unique to the individual.

These fragments are called polymorphisms be- cause they vary in shape from person to person. ✄ DNA profiling is the process of separating an individual's unique poly-

morphic fragments from the common ones to identify one individual.

DNA profiling

DNA profiling can be used to determine paternity and also in forensic investigations. Match polymorphisms.

DNA profiling

Profile obtained from bloodleft at scene of an assault.Unique fragments of DNA are compared.

Profile obtained from convictedoffender and entered in CODIS*

*Combined DNA Index System – a computerized data base of offenders

DNA profiling can be used to determine paternity, and also in forensic investigations. Social implications: ✄ Identity issues for children who discover a biological father is different from whom was expected. ✄ Fatherhood issues for an unmarried teen – when a

guy learns he is or is not the father. ✄ When responsibility

for a crime is con- firmed or denied

decades after the crime was commit-

ted.

DNA profiling

The human genome has recently been completely decod- ed (or sequenced). The genome is the set of all the genes that produce a human being. ✄ As we identify the similarities - and the differences - among the genes of mammals and other organisms, we will gain

valuable new insights into human evo-

lution.

Sequencing the human genome

The human genome has recently been completely decod- ed (or sequenced). The genome is the set of all the genes that produce a human being. ✄ The genetic code can be used to identify disease-

associated genes and eventually indicators of

what drugs can be effective against the

illness.

Sequencing the human genome

The human genome has recently been completely decod- ed (or sequenced). The genome is the set of all the genes that produce a human being. ✄ Parents can possibly choose to select a baby’s char- acteristics (as in the movie GATTACA): improved

physical stamina and lifespan, ath-

leticism, height, hair color, etc.

Sequencing the human genome

Genetic Engineering and Biotechnology

When genes are transferred between species, the amino acid sequence of polypeptides translated from them is unchanged because the genetic code is universal. ✄ DNA triplets code for

the same amino acids in every organism.

This supports the theory of evolution: one common ancestor.

Universal genetic code

DNA technology makes it possible to clone genes. a) A foreign gene is inserted into a bacterial plasmid, producing recombinant DNA - artificial DNA from 2 sources.

b) This recombinant plasmid is returned to a bacterial cell. Every time this cell reproduces, the recombin-

ant plasmid is replicated as well (cloned) and passed

on to its descendents. ✄ Clone – an identical copy of the original.

Gene transfer techniques

Plasmid

Transformation

Overview

Restriction enzymes are used to make recombinant DNA. ✄ Restriction enzymes cut DNA molecules at specific locations. Ex: EcoR1 cuts at

3'-CTTAAG- 5', wherever it occurs.

✄ They cut in staggered way creating single-stranded “sticky” ends. These will

form hydrogen bonds with complementary sin- gle-stranded stretches on other DNA molecules.

Gene transfer techniques

Cloning a gene into a bacterial plasmid requires 5 steps. 1) Isolate the plasmid and the foreign gene. ✄ Plasmids are small circular bits of extra

DNA found in bacteria that often conf- er resistance to toxins in environments.

✄ A variety of artificial plasmid vectors have been construct- ed for this purpose with special sites for restriction

enzymes and resistance to antibiotics.

Gene transfer techniques

The “gene of interest” is the foreign DNA to be inserted into the host.

Cloning a gene into a bacterial plasmid requires 5 steps. 2) Use a restriction enzyme to cut out the gene and to open the plasmid (producing sticky ends on each), mix them, then use DNA ligase to splice the sticky ends together.

Gene transfer techniques

Cloning a gene into a bacterial plasmid requires 5 steps. 3) Put the recombinant plasmid into bacteria (the process of transformation – changing an organism by adding foreign DNA). ✄ Mix the plasmid DNA and the bacteria under the proper

conditions, and a small amount of DNA will pass through

the bacterial cell wall.

Gene transfer techniques

Cloning a gene into a bacterial plasmid requires 5 steps. 4) Clone the cells - use a growth medium containing the selected antibiotic. ✄ Only cells with the antibiotic resistance (on the plasmid)

can grow. Instead of looking at millions of colonies, only a few will grow on the culture medium.

5) Identify the clones.

Gene transfer techniques

Resistant cells should contain the foreign DNA.

Current uses of genetic modification techniques: ✄ Grow GM bacteria for production of proteins. ✄ Transfer genes into other organisms.

GM crops and animals

Note the applications

Genetically modified crops Benefits: ✄ Plants could make their own nitrogen fertilizer by adding genes from nitrogen-fixing bacteria that make NH4 from N2 in the atmosphere. ✄ Resistance to an herbicide (weed killer) called glyphosate

was brought into cotton and other plants from bacteria. ✄ Resistance to insects – a bacterial protein that kills insects

was used to transform crop plants. If insects (like cater- pillars) eat the plant they die, but humans are not affected. + All of these uses would

save farmers money and reduce environmentalpollution.

GM crops and animals

Genetically modified crops Hazards: ✄ Introduced genes could “escape” into the environment and have unintended effects:

- Weeds could acquire the gene and become “super weeds”.- Pollinators (bees and butterflies) could die as well as the pests,

or herbivores could be affected. ✄ Humans could be affected (allergies)

- Many European governments will not accept American plant pro-

ducts for fear that they are GM (economic issues) with unknown

health risks.

GM crops and animals

Genetically modified crops Ethics of use: ✄ Should humans be changing

other creatures just to suit our purposes?

✄ How do we assess the risks and avert a disaster?

✄ Who is responsible when something goes wrong? Who cleans up the mess?

✄ In Europe some people take it upon themselves to de-stroy fields of GM crops.

GM crops and animals

Definition: Clone – a group of genetically identical organisms, or a group of cells derived from a single parent cell.

Cloning animal cells

A technique for cloning using differentiated animal cells

Reasons for cloning animals: ✄ Produce a group of horses, cows, etc. with superior

qualities. ✄ “Bring back” beloved pets. ✄ Bring extinct animals back to

life. Ex: the Tasmanian wolf, hunted to extinction >100 years ago.

Cloning animal cells

Dolly, the ewe:the cloned cells weretaken from her udder.

Definition: Therapeutic cloning – the creation of an embryo to supply embryonic stem cells for medical use. ✄ A human embryo

is produced and destroyed. Is that murder?

✄ A life can be saved when new organs are produced in this way, and there are no problems with rejection.

Therapeutic cloning