DNA Technology and Genomics. History of DNA Technology and Genetic Engineering Genetic Engineering is the process of manipulating genes and genomes. 1953.

DNA Technology and Genomics

History of DNA Technology and Genetic Engineering

• Genetic Engineering is the process of manipulating genes and genomes.

• 1953 Watson, Crick, Wilkins and Franklin’s discovery of the DNA double-helix

• By 2003 the entire human genome had been sequenced by Craig Venter and the Human Genome Project, years ahead of schedule.

• But the roots of DNA technology stretch back to the dawn of history…

Selective breeding• Since the days that humans settled in the fertile river

valleys, farmers have been trying to perfect crops.• Selective breeding is the process of choosing

organisms with different desirable traits and mating them to produce “improved” offspring.

• Black angus beef, Chihuahuas, and bananas are all examples of selective breeding; over generations, individuals with a set of characteristics are produced.

Selective Breeding and Polyploidy• In plants, one way to cram in all of the

desired traits is to combine all of the parents’ chromosomes.

• So instead of having 2 copies of each chromosome, some plants have 4, 6, or 8 copies of each chromosome.

• We call species with more than 2 copies of each chromosome polyploidy.

• Most of our food crops are polyploid, and most polyploidy organisms are sterile.

• What are some possible disadvantages?

Genetic Engineering and Recombinant DNA

• Genetic engineering involves using bacteria like scissors, to cut sections of DNA, and then gluing in new genes that come from a different species.

• The DNA that has been artificially made is called recombinant DNA.

• The product of genetic engineering is a transgenic organism.• This process is still controversial, with some countries requiring

food products to be labeled GM, or “genetically modified” if they contain ingredients from a transgenic organism.

• Example: Herbicide tolerance in corn, soybeans, and cotton

Cloning• Gene cloning is the process by which scientists can produce

multiple copies of specific segments of DNA that they can then work with in the lab

• Restriction enzymes are used to cut strands of DNA at specific locations (called restriction sites). They are derived from bacteria.

• When a DNA molecule is cut by restriction enzymes, the result will always be a set of restriction fragments, which will have at least one single-stranded end, called a sticky end.

• Sticky ends can form hydrogen bonds with complementary single-stranded pieces of DNA. These unions can be sealed with enzyme DNA ligase

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Gene Cloning

Use of Cloning• There are two distinct branches of cloning. • Cell cloning involves inserting a patient’s DNA into an

“adult” cell and growing a tissue or organ in culture.• This kind of cloning is widespread and has medical

applications from organ transplants, to skin grafts for burn victims, to neural tissue for muscular dystrophy sufferers.

• Organism cloning involves putting DNA into an “embryonic” cell, or stem cell, which could still be any kind of body part, and then incubating the growing organism.

• This practice has been going on for years with plants by “taking cuttings” but only recently in animals, with Masha the mouse, born the first mammalian clone in 1986.

Cloning a Gene in a Bacterial Plasmid

Finding A Gene of Interest• Nucleic Acid hybridization is used to find genes of

interest after transformation.– If we know part of the nucleotide sequence of the gene

we want, we can synthesize a probe complementary to it.• G-G-C-T-A-A• Probe: C-C-G-A-T-T

– We now have genomic libraries that log sets of thousands of recombinant plasmid clones, each of which has a piece of the original genome being studied

– A cDNA library is made up of complementary DNA made from mRNA transcribed by reverse transcriptase

Polymerase chain reaction (PCR)

• Amplification of any piece of DNA without cells (in vitro)

• Materials: heat, DNA polymerase, nucleotides, single-stranded DNA primers

• Applications: fossils, forensics, prenatal diagnosis, etc.

DNA Technology and Forensics• CSI and other crime shows, as well as the

paternity tests popular in talk shows, depend on a process called gel electrophoresis.

• Gel electrophoresis involves running an electrical current through a gelatin to separate DNA into bands.– How does the current help separate?

This DNA has been cut by a restriction enzyme, creating a kind of DNA signature.

A DNA Diagnosis• DNA microarrays, microchips printed with DNA, show

which genes are producing proteins• These have the potential to change medicine, as they

can show which genes are functioning properly or not at all and diagnose a disease or disorder years before it becomes a threat.

Gene Linkage Mapping• Our first close look at the sequence of DNA was

through gene linkage mapping.– Gene linkage mapping measure the distance between

genes by the frequency of crossing over moving one to the homologous chromosome.

– Distant genes are separated by crossing over more often than nearby genes.

Karyotyping• A karyotype is a picture of an organism’s chromosomes.• This is useful in medicine because a karyotype can be

used to detect genetic abnormalities, like duplicate or fragmented chromosomes.

DNA sequencing and The Human Genome Project

• After they knew the order of genes, scientists became curious about the order of nucleotides in these genes, or gene sequence.

• The first organism to be sequenced was a bacteriaphage in 1975.

• In 1986, the US Department of Energy announced its intention to sequence all human genes, also called the human genome.

• This was formalized in 1990 as the Human Genome project, a race to sequence all human genes.

Human Genome Project• In 1990, scientists guessed there were between

50,000 and 100,00 genes in the Human Genome. By 2001, the number had dropped to about 35,000 genes.

• When the project was completed in 2003, 2 years ahead of schedule, the final tally came to 19,599, far less than expected.

• Going on complexity alone, humans should have more genes, C. elegans, one of the simplest animals with only a few hundred cells has about 20,000 genes.

• Even bacteria have about 5,000 genes on their one DNA molecule!!

Why So Few?

• Well, we have already learned that not every RNA codon codes for a different amino acid – CCA, CCG, CCU, and CCC all call for proline

• Apparently, gene number is not related to how complex an organism is.

• RNA editing, protein folding, multiple roles of 1 protein, and other factors must account for the complexity we see.

• So now the new race is to sequence the human proteome, or all the proteins humans make.

• How can knowing the genome and proteome help advance medicine?

Bacterial Transformation

Review of the Historical Experiments Fred Griffith

Avery, MacLeod , McCarty

Fred Griffith 1928

• Studied different strains of Streptococcus pneumoniae, the bacteria that causes pneumonia.

• This bacteria come in two strains: S and R• S-form

– capsule and looks smooth under the microscope. – Virulent and kills infected mice because the

immune system cannot break through the cell wall.

• The R-form – doesn't have a capsule and appears rough.– The immune system is able to destroy the cell wall.– This makes the R form non-virulent. The mice

infected by this form of the bacteria will survive.

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Griffith's Experiments

• Griffith injected mice with the S form of bacteria and all the mice died from pneumonia.

• He injected mice with the R form of bacteria and the mice survived the infection.

• He then killed the S form by exposing them to high temperatures. Mice injected with these heat-killed bacteria survived with no ill effects.

• He mixed his heat-killed, disease-causing bacteria with live, harmless ones and injected the mixture into mice.

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Griffith's Experiments

• He expected the mice to survive because both strains were harmless. But, the mice died from pneumonia!!!

• And he found living S cells in the mice! • Somehow the heat-killed S strain passed their

ability to cause disease to the live R strain. • Griffith called this Transformation

– one strain of bacteria had been changed into another. Some factor was transferred from heat-killed cell into the live cells. He also found that the change was permanent.

• He hypothesized that factor could be a gene that could change the properties of bacteria. 22

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Frederick Griffith 1928Transformation of Bacteria

Avery, MacLeod and McCarty 1944

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Avery and his colleagues decided to expand on Griffith’s experiments to try to identify the “transforming” material.

Separated the Components

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• Ruptured heat killed S strain bacteria to release their contents.

• Separated and purified the RNA, DNA, proteins and the polysaccharide capsules from the bacteria into separate factions.

• Mixed each faction with live R bacterial cells and injected them into mice.– R-bacteria + RNA from S-bacteria = live mouse– R-bacteria + proteins from S-bacteria = live mouse– R-bacteria + polysaccharides from S-bacteria = live mouse

Only R cells were found in their blood.

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Separated the Components• Ruptured heat killed S strain bacteria to release

their contents.• Separated and purified the RNA, DNA, proteins

and the polysaccharide capsules from the bacteria into separate factions.

• Mixed each faction with live R bacterial cells and injected them into mice.– R-bacteria + RNA from S-bacteria = live mouse– R-bacteria + proteins from S-bacteria = live

mouse– R-bacteria + polysaccharides from S-bacteria

= live mouse– Only R cells were found in their blood.– R bacteria + DNA from S-bacteria = DEAD

MOUSE!!!– They concluded that DNA was the hereditary

material

Science is full of surprises… be careful with “no way!”

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Despite the fact that the Transforming substance had to be resistant to heat, and proteins are inactivated by heat…

Most scientists thought that proteins were the hereditary material because they were more complex and varied than nucleic acids.

Scientists were generally skeptical, believing DNA to be too simple a molecule to contain all the genetic information for an organism.