Recombinant DNA and Genetic Engineering

Chapter 16

Impacts, Issues:

Golden Rice or Frankenfood?

Scientists created transgenic rice (Golden Rice) as a vitamin A supplement for undernourished nations; is the benefit worth the risk in these gene-manipulated food sources?

16.1 Cloning DNA

Process to add genes to food or other cell types is simple in principle Researchers cut up DNA from different sources, then paste the resulting fragments together Cloning vectors can carry foreign DNA into host cells

Cut and Paste for New DNA Combos

Restriction enzymes
Bacterial enzymes that cut DNA wherever a specific nucleotide sequence occurs

Single-stranded DNA tails produced by the same restriction enzyme base-pair together
DNA ligase bonds sticky ends together

Recombinant DNA
Composed of DNA from two or more organisms

Making Recombinant DNA

This is especially useful to introduce genes into a sequence in research.

DNA Cloning

DNA cut into fragments by restriction enzymes is inserted into cloning vectors (plasmids) cut with the same enzyme Cloning vectors with foreign DNA are placed in host cells, which divide and produce many clones, each with a copy of the foreign DNA

Cloning Vectors

DNA Cloning

cDNA Cloning
Complementary DNA (cDNA)
DNA made from an mRNA template

Reverse transcriptase transcribes mRNA to DNA, forming a hybrid molecule

DNA polymerase builds a double-stranded DNA molecule that can be cloned Especially useful to obtain DNA without introns.

cDNA Cloning by Reverse Transcriptase

16.1 Key Concepts

DNA Cloning by Lab and Plasmids

Researchers routinely make recombinant DNA by cutting and pasting together DNA from different species Plasmids and other vectors can carry foreign DNA into host cells

Genomes and DNA Libraries

Genome
The entire set of genetic material of an organism

DNA libraries are sets of cells containing various cloned DNA fragments
Genomic libraries (all DNA in a genome) cDNA libraries (all active genes in a cell)

Probes Used for ID of DNA

Probe
A fragment of DNA labeled with a tracer Used to find a specific clone carrying DNA of interest in a library of many clones

Nucleic acid hybridization

Base pairing between DNA from different sources A probe hybridizes with the targeted gene

Big-Time DNA Amplification: PCR

Polymerase chain reaction (PCR)

A cycled reaction that uses a heat-tolerant form of DNA polymerase (Taq polymerase) to produce billions of copies of a DNA fragment This is how a single drop of blood at a crime scene can become expanded to enough to make necessary tests and still be available for future testing if needed

PCR in Overview

DNA to be copied is mixed with DNA polymerase, nucleotides and primers that basepair with certain DNA sequences Cycles of high and low temperatures break and reform hydrogen bonds between DNA strands, doubling the amount of DNA in each cycle

PCR Steps

to More

16.2 Key Concepts

Needles in Haystacks

Researchers manipulate targeted genes by isolating and making many copies of particular DNA fragments

16.3 DNA Sequencing

DNA sequencing reveals the order of nucleotide bases in a fragment of DNA

DNA Sequencing
DNA is synthesized with normal nucleotides and dideoxynucleotides tagged with different colors
When a tagged base is added, DNA synthesis stops; fragments of all lengths are made

Electrophoresis separates the fragments of DNA, each ending with a tagged base, by length
Order of colored bases is the sequence of DNA Finished sequence is basis for comparison

DNA Sequencing

16.4 DNA Fingerprinting

One individual can be distinguished from all others on the basis of DNA fingerprints Confidence here in results is extremely high, in the usually stated range of one in many millions

DNA Fingerprints
DNA fingerprint
A unique array of DNA sequences used to identify individuals

Short tandem repeats (STRs)

Many copies of the same 2- to 10-base-pair sequences in a series along a chromosome Types and numbers of STRs vary greatly among individuals

Creating DNA Fingerprints

PCR is used to amplify DNA from regions of several chromosomes that have STRs Electrophoresis is used to separate the fragments and create a unique DNA fingerprint DNA fingerprints have many applications
Legal cases, forensics, population studies

DNA Fingerprints: Forensics Case Example

You are on the jury. You are shown this prepared comparison of DNA fingerprints, with ID as shown. See if you can match suspect with sample from the crime scene.

16.3-16.4 Key Concepts

Deciphering DNA Fragments

Sequencing reveals the linear order of nucleotides in a fragment of DNA A DNA fingerprint is an individuals unique array of DNA sequences

16.5 Studying Genomes

Comparing the sequence of our genome with that of other species is giving us insights into how the human body works You already know of 98 percent same human sequences with that of chimpanzes How about 49 percent the same between a banana and a human?

The Human Genome Project

Automated DNA sequencing and PCR allowed human genome projects to sequence the 3 billion bases in the human genome 28,976 genes have been identified, but not all of their products or functions are known As of 2010, distinct gene numbers down to about 23,000 by best estimates from work

Sequencing the Human Genome

Computers have greatly speeded process up and also increased accuracy.

Genomics is a Growing Application

Genomics: The study of genomes
Structural genomics Comparative genomics

Analysis of the human genome yields new information about genes and how they work
Applications in medicine and other fields Example: APOA5 mutations and triglycerides

DNA Chips Have a Future

DNA chips
Microarrays of many different DNA samples arranged on a glass plate Used to compare patterns of gene expression among cells of different types or under different conditions May be used to screen for genetic abnormalities, pathogens, or cancer

16.6 Genetic Engineering

Genetic engineering
A laboratory process by which deliberate changes are introduced into an individuals genome

Todays most common genetically modified organisms are bacteria and yeast
Are used in research, medicine, and industry Example: production of human insulin

GMOs Now and Later

Genetically modified organisms (GMOs)
Individuals containing modified genes from the same species or a different species Future will have major control problems as the developer of GMO usually patents process/result

Transgenic organisms
Individuals containing genes transferred from a different species (also GMOs) Example: Bacteria with jellyfish genes

16.7 Designer Plants by GM

Genetically engineered crop plants are widespread in the United States But can their designed change(s) jump to other plant life or end up incorporated in animals eating the modified plants?

The Ti Plasmid a GMO Mechanism

Ti plasmid
Plasmid of bacteria Agrobacterium tumefaciens Contains tumor-inducing (Ti) genes Used as a vector to transfer foreign or modified genes into plants, including some food crops

Ti Plasmid Transfer Steps

Genetically Engineered Plants

Crop plants are genetically modified to produce more food at lower cost
Resistance to disease or herbicides Increased yield Plants that make pesticides (Bt protein gene) Drought resistance

GMO Controversies
73 GMO crops are approved for use in US, with hundreds more pending
Corn, sorghum, cotton, soy, canola, alfalfa Big problem of just a few companies doing nearly all the research and manufacturing can lead to a monopoly problem in future

Facts and controversy real life

In crops engineered for herbicide resistance, weeds are becoming resistant to herbicides Engineered genes are spreading into wild plants and nonengineered crops

Some Genetically Modified Plants

16.8 Biotech Barnyards

Animals that would be impossible to produce by traditional breeding methods are being created by genetic engineering This can be really good for endangered animals Genetically engineered animals are used in research, medicine, and industry

Of Mice and Men

1982: The first transgenic animals mice with genes for rat growth hormone

Examples of Transgenic Animals

Genetically modified animals are used as models of many human diseases
Mice used in knockout experiments

Genetically modified animals make proteins with medical and industrial applications
Goats and rabbits that make human proteins Farms animals with desirable characteristics

Some Genetically Modified Animals

That silly-looking featherless chicken is easily the most commercially viable possibility shown here. It would eliminate a costly part of chicken processing and could enable very warm climate poultry farms.

Knockout Cells and Organ Factories

Transgenic pigs with human proteins are a potential source of organs and tissues for transplants in humans
May prevent rejection by immune system

Xenotransplantation
Transplantation of a tissue or organ from one species to another Pig heart valves used for many years.

16.10 Modified Humans?

We as a society continue to work our way through the ethical implications of applying new DNA technologies The manipulation of individual genomes continues even as we are weighing the risks and benefits of this research

Gene Therapy Helping the Individual

Gene therapy
Transfer of recombinant DNA into body cells to correct a genetic defect or treat a disease Viral vectors or lipid clusters insert an unmutated gene into an individuals chromosomes Examples: Cystic fibrosis, SCID-X1

Getting Better by Gene Therapy

1998: A viral vector was used to insert unmutated IL2RG genes into boys with severe combined immunodeficiency disease (SCID-X1) most recovered immune function

Getting Worse by Gene Therapy

No one can predict where a virus-injected gene will insert into a chromosome several boys from the SCID-X1 study developed cancer In other studies, severe allergic reactions to the viral vector itself have resulted in death

Getting Perfect Over Time

Eugenic engineering
Engineering humans for particular desirable traits, not associated with treatment of disorders

16.6-16.10 Key Concepts

Using the New Technologies of GM

Genetic engineering, the directed modification of an organisms genes, is now used in research, and it is being tested in medical applications Many questions must be answered about the ethics and consequences of manipulating the human genome some of these can be answered by our government but many will remain answerable only by the individual as he or she agrees to their usage personally.