Genetic Technology

Genetic Engineering

• Deliberately altering the information content of DNA molecules

• Genes are isolated, modified, and inserted into an organism

• Made possible by recombinant technology

– Cut DNA up and recombine pieces

– Amplify modified pieces

Gene Therapy

• The transfer of one or more normal or

modified genes into and individual’s

body cells to correct a genetic defect or

boost resistance to disease

Restriction Enzymes

• Hamilton Smith was studying how Haemophilus influenzae defend themselves from bacteriophage attack– Discovered bacteria have an enzyme that chops up

viral DNA

• Restriction enzymes cut DNA at a specific sequence

• Number of cuts made in DNA will depend on number of times the “target” sequence occurs

• Recombinant DNA can be formed: by splicing 2 different segments of DNA together to create a new strand

Making Recombinant DNA

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one DNA fragment another DNA fragment

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In-text figurePage 254

•Recombinant DNA can be formed: by splicing 2 different segments of DNA together to create a new strand

Making Recombinant DNA

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DNA ligase action

In-text figurePage 254

Using Plasmids

• Plasmid is small circle of bacterial DNA

• Foreign DNA can be inserted into

plasmid

– Forms recombinant plasmids

– Plasmid is a cloning vector

– Can deliver DNA into another cell

Using Plasmids

DNA fragments+enzymes

recombinantplasmids

host cells containing recombinant plasmids

Figure 16.4Page 255

Making cDNAmRNA transcript

mRNA–cDNA hybrid

single-stranded cDNA

double-stranded cDNAFigure 16.5Page 255

•cDNA is a DNA molecule copied from a mature mRNA transcript by reverse transcription

Amplifying DNA

• Ways to take small pieces of DNA and make them larger

– Fragments can be inserted into fast-growing microorganisms

– Polymerase chain reaction (PCR)

Polymerase Chain Reaction

• Sequence to be copied is heated

• Primers are added and bind to ends of single strands

• DNA polymerase uses free nucleotides to create complementary strands

• Doubles number of copies of DNA

Polymerase Chain Reaction

Double-stranded DNA to copy

DNA heated to 90°– 94°C

Primers added to base-pair with ends

Mixture cooled; base-pairing of primers and ends of DNA strands

DNA polymerasesassemble new DNA strands

Figure 16.6Page 256

Stepped Art

Polymerase Chain Reaction

Figure 16.6Page 256

Stepped Art

Mixture heated again; makes all DNA fragments unwind

Mixture cooled; base-pairing between primers and ends of single DNA strands

DNA polymerase action again doubles number of identical DNA fragments

DNA Fingerprints

• Individuals have a unique array of DNA

fragments

• Inherited from parents in Mendelian

fashion

• Even full siblings can be distinguished

from one another by this technique

Tandem Repeats

• Short regions of DNA that differ substantially among people

• Many sites in genome where tandem repeats occur

• Each person carries a unique combination of repeat numbers

RFLPs

• Restriction fragment length polymorphisms

• DNA from areas with tandem repeats is cut with restriction enzymes

• Because of the variation in the amount of repeated DNA, the restriction fragments vary in size

• Variation is detected by gel electrophoresis

Gel Electrophoresis

• DNA is placed at one end of a gel

• A current is applied to the gel

• DNA molecules are negatively charged and move toward positive end of gel

• Smaller molecules move faster than larger ones

Analyzing DNA Fingerprints

• DNA is stained or made visible by use of

a radioactive probe

• Pattern of bands is used to:

– Identify or rule out criminal suspects

– Identify bodies

– Determine paternity

Genome Sequencing

• 1995 - Sequence of bacterium Haemophilus influenzae determined

• Automated DNA sequencing now main method

• Draft sequence of entire human genome determined in this way

• Human Genome Project

The Human Genome Initiative

Goal - Map the entire human genome• Initially thought by many to be a waste

of resources• Process accelerated when Craig

Ventner used bits of cDNAs as hooks to find genes

• Sequencing was completed ahead of schedule in early 2001

Genomics

• Structural genomics: actual mapping and sequencing of genomes of individuals

• Comparative genomics: concerned with possible evolutionary relationships of groups of organisms

Gene Libraries

• Bacteria that contain different

cloned DNA fragments

– Genomic library

– cDNA library

Using a Probe to Find a Gene

• You want to find which bacteria in

a library contain a specific gene

• Need a probe for that gene

– A radioisotope-labeled piece of

DNA

– Will base-pair with gene of

interest

Colonies on plate

Cells adhere to filter

Cells are lysed;DNA sticks to filter

Probe is added

dark spot on film, indicates colony with gene

Location where probe binds forms

Cloning

•Making a genetically identical copy of DNA or of an organism

•Three different types–Recombinant DNA or DNA cloning (we already talked about)–Reproductive Cloning – making of an entire organism–Therapeutic Cloning – also called “embryo cloning”; stem cells

Cloning Dolly

1997 - A sheep cloned from an adult cell

– Nucleus from mammary gland cell was

inserted into enucleated egg

– Embryo implanted into surrogate mother

– Sheep is genetic replica of animal from

which mammary cell was taken

Engineered Proteins

• Bacteria can be used to grow medically

valuable proteins

– Insulin, interferon, blood-clotting

factors

– Vaccines

Cleaning Up the Environment

• Microorganisms normally break down

organic wastes and cycle materials

• Some can be engineered to break down

pollutants or to take up larger amounts

of harmful materials

Engineered Plants

• Cotton plants that display resistance to herbicide

• Aspen plants that produce less lignin and more cellulose

• Tobacco plants that produce human proteins

• Mustard plant cells that produce biodegradable plastic

First Engineered Mammals

• Experimenters used mice with hormone deficiency that leads to dwarfism

• Fertilized mouse eggs were injected with gene for rat growth hormone

• Gene was integrated into mouse DNA

• Engineered mice were 1-1/2 times larger than unmodified littermates

Can Genetically Engineered Bacteria “Escape”?

• Genetically engineered bacteria are designed so that they cannot survive outside lab

• Genes are included that will be turned on in outside environment, triggering death

Ethical Issues

• Who decides what should be “corrected”

through genetic engineering?

• Should animals be modified to provide

organs for human transplants?

• Should humans be cloned?