Biotechnology

Chapter 20Biotechnology What is Cloning?Cloning is the creation of an organism that is an exact genetic copy of another. This means that every single bit of DNA is the same between the two!

What animals have been cloned?

In 1952, the first animal, a tadpole, was cloned. Before the creation of Dolly, the first mammal cloned from the cell of an adult animal, clones were created from embryonic cells. Since Dolly, researchers have cloned:Sheep, goats, cows, mice, pigs, cats, rabbits, and a gaur. All these clones were created using nuclear transfer technology. Some species may be more resistant to somatic cell nuclear transfer than others. Traumatic experience , and improvements in cloning technologies may be needed before many species can be cloned successfully.

Types of CloningSomatic Cell Nuclear Transfer(Reproductive cloning)Recombinant DNA Technology or DNA CloningTherapeutic cloning

Somatic Cell Nuclear Transfer (SCNT)

Produces an exact clone, or genetic copy, of an individual. Method used to create Dolly the Sheep.Somatic cell: Any cell in the body other than the gametes. In mammals, every somatic cell has two complete sets of chromosomes (diploid)

Somatic Cell Nuclear Transfer (SCNT) & Dolly (Reproductive Cloning )

Researchers isolated a somatic cell from an adult female sheep. Next, they transferred the nucleus from that cell to an egg cell from which the nucleus had been removed. The egg cell, with its new nucleus, was behaving just like a fertilized zygote. It developed into an embryo, which was implanted into a surrogate mother and carried to term.

Creating Dolly

Stage 1

Cell collected from a sheeps udder.Stage 2

Nucleus is removed from unfertilized egg of second sheep.Stage 3

Udder cell is inserted into egg with no nucleus.Stage 4

Insertion is successful.Stage 5

Electrical charge is supplied.Stage 6

Cells begin to divide.Stage 7Embryo implanted into a surrogate

Celebrity Sheep Died at Age 6Dolly was put down by lethal injection Feb. 14, 2003. Prior to her death, Dolly had been suffering from lung cancer and crippling arthritis. Although most Finn Dorset sheep live to be 11 to 12 years of age, postmortem examination of Dolly seemed to indicate that, other than her cancer and arthritis, she appeared to be quite normal. The unnamed sheep from which Dolly was cloned had died several years prior to her creation. Dolly was a mother to six lambs, bred the old-fashioned way.

Is cloning an organism the same as cloning a gene?

Cloning an animal, or any other organism, refers to making an exact genetic copy of that organism. Cloning a gene means isolating an exact copy of a single gene from the entire genome of an organism. Involves copying the DNA sequence of that gene into a smaller, more accessible piece of DNA, such as a plasmid. This makes it easier to study the function of the individual gene in the laboratory.Animation

Recombinant DNA Technology or DNA CloningTransfer of a DNA fragment from one organism to a self-replicating genetic element such as a bacterial plasmid. The DNA is then be propagated in a foreign host cell. Been around since the 1970s, and it has become a common practice in molecular biology labs today.

PlasmidsSelf-replicating extra-chromosomal circular DNA molecules Used by Human Genome Project (HGP) researchers to copy genes

To "clone a gene"A DNA fragment containing the gene of interest is isolated from chromosomal DNA using restriction enzymes and then united with a plasmid that has been cut with the same restriction enzymes. When the fragment of chromosomal DNA is joined with its cloning vector in the lab, it is called a "recombinant DNA molecule." Following introduction into suitable host cells, the recombinant DNA can then be reproduced along with the host cell DNA.

Restriction Enzymes

Aka: restriction endonucleasesDNA-cutting enzymes Found in bacteria & made exclusively by prokaryotes.

In order to sequence DNA, it is first necessary to cut it into smaller fragments. A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides.Originally discovered through their ability to break down, or restrict, foreign DNA. Can distinguish between the DNA normally present in the cell and foreign DNAIe. infecting bacteria virus DNA (bacteriophage). Defend the cell from invasion by cutting the foreign DNA into pieces, thereby rendering the DNA nonfunctional.

Restriction Enzymes

Many restriction enzymes cut in an offset fashion. The ends of the cut have an overhanging piece of single-stranded DNA. Known as "sticky ends" They are able to form base pairs with any DNA molecule that contains the complementary sticky end. The union can be made permanent by DNA ligase, that forms covalent bonds along the backbone of each strand. The result is a molecule of recombinant DNA (rDNA). Animation

Restriction Enzymes & Nucleases

Nuclease -any enzyme that cuts the phosphodiester bonds of the DNA backbonebond between a two sugar groups and a phosphate group

Endonuclease -an enzyme that cuts some where within a DNA molecule.

Exonuclease- cuts phosphodiester bonds by starting from the free end of the a single DNA strand in the 3' to 5' direction

Restriction Enzymes

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Palindrome DNAA sequence of DNA which reads the same on both strands Ie. a palindrome). Such sequences often form the recognition sites for enzymes such as restriction endonucleases.

Restriction Enzyme EcoR1EcoRI, one of many restriction enzymes, is obtained from the bacteria Escherichia coli.

Benefits of Recombinant DNA Revolutionized genetics & biotechnology industryExamples:Human insulin Human factor VIII (for males with hemophilia A),

Other Cloning Vectors Bacterial plasmidsVirusesBacteria artificial chromosomes (BACs)Yeast artificial chromosomes (YACs)Cosmids are artificially constructed cloning vectors for infection into E. coli cells. Bacteria are most often used as the host cells for recombinant DNA molecules, but yeast and mammalian cells also are used.

Therapeutic Cloning

Aka: "embryo cloning Production of human embryos for use in research. Goal is not to create cloned human beings, but rather to harvest stem cells that can be used to study human development and to treat disease. Animation

Can organs be cloned for use in transplants?

To do this, DNA would be extracted from the person in need of a transplant and inserted into an enucleated egg. After the egg containing the patient's DNA starts to divide, embryonic stem cells that can be transformed into any type of tissue would be harvested. The stem cells would be used to generate an organ or tissue that is a genetic match to the recipient. In theory, the cloned organ could then be transplanted into the patient without the risk of tissue rejection.

Many challenges must be overcome before "cloned organ" transplants become reality.More effective technologies for creating human embryos, harvesting stem cells, and producing organs from stem cells would have to be developed. In 2001, scientists cloned the first human embryos; however, the only embryo to survive the cloning process stopped developing after dividing into six cells. In 2002, scientists successfully transplanted kidney-like organs into cows. The team of researchers created a cloned cow embryo by removing the DNA from an egg cell and then injecting the DNA from the skin cell of the donor cow's ear. Since little is known about manipulating embryonic stem cells from cows, the scientists let the cloned embryos develop into fetuses. The scientists then harvested fetal tissue from the clones and transplanted it into the donor cow. In the three months of observation following the transplant, no sign of immune rejection was observed in the transplant recipient.

What are the risks of cloning?

Reproductive cloning is expensive and highly inefficient. More than 90% of cloning attempts fail to produce viable offspring. More than 100 nuclear transfer procedures could be required to produce one viable clone. Cloned animals tend to have more compromised immune function Clones have been known to die mysteriously.Example, Australia's first cloned sheep appeared healthy and energetic on the day she died, and the results from her autopsy failed to determine a cause of death.

More Risks Programming errors in the genetic material from a donor cell. When an embryo is created from the union of a sperm and an egg, the embryo receives copies of most genes from both parents. A process called "imprinting" chemically marks the DNA from the mother and father so that only one copy of a gene (either the maternal or paternal gene) is turned on. Defects in the genetic imprint of DNA from a single donor cell may lead to some of the developmental abnormalities of cloned embryosPCRPurpose: Used to identify and amplify (copy)DNA in a sample Makes millions of copies of a geneNecessary to have enough starting template for sequencing.BenefitsTakes only approximately 2 hoursBypasses the need to use bacteria for amplifying DNA.Relatively simple and inexpensive Used every day to diagnose diseases, identify bacteria and viruses, match criminals to crime scenes, and in many other waysAnimation.Animation

Steps of PCRPair of primers (short single-stranded DNA segments) are needed that attach to either side of the target DNA to be copied. The target DNA, the primers, a polymerase molecule (heat resistant)which duplicates DNA, and a supply of nucleotides are mixed together are go through a series of heating and cooling cycles. After approximately 30 cycles, the segment will have been copied 250 million times. The results of PCR can then be analyzed in various ways, such as agarose gel electrophoresis or sequencing.

Enzymes & Primers Primers:About 20 nucleotides longComplementary to the beginning and end of the DNA fragment of interest which one needs to amplify. Select the boundaries of the region to be amplified by PCR. During the PCR annealing cycle, PCR primers anneal to the complementary region of the DNA. Anneal-to heat and then cool in order to separate strands and induce combination at lower temperatures especially with complementary strands

Heating & Cooling Process1st Heating ProcessSamples heated to 95 degrees C

Purpose: Denature DNA- breaking bonds

2nd Cooling Process (Annealing)60 degrees C

Allows primers to form Hydrogen bonds or anneals with complementary DNA strand

Heating & Cooling Process3rd Heating ProcessTemperature raised to 72 degrees CTaq polymerase(optimal temperature) begins polymerization adding nucleotides to the 3end of each primer attached to a DNA strand

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The segment of the target DNA between the primers will be copied during each cycle, accumulating in the mixture in an exponential fashion.

After 1st complete cycle-----2 double stranded copies of the target DNAAfter 2nd complete cycle----- 4 copiesAfter 3rd complete cycle----- 8 copiesAfter 25 cycles- 33 million copies

Steps of PCR

PCR

Steps of PCR

Enzymes & Primers Taq polymeraseObtained from hot spring bacteriaAble to withstand extreme PCR heat

Gel electrophoresis.When restrictive endonuclease is used the DNA split to multiple mixed fragments, they are then separated by gel electrophoresis. The gel is then blotted on nitrocellulose paper. Turns to a solid form Now we have separate solid DNA fragments. A probe is then added to bind to its matching fragment which can then be visualized by autoradiography.Animation

Types of Blotting If the substance is DNA it is termed Southern Blotting.If the substance is RNA it is termed Northern blotting.If the substance is protein it is termed Western blotting.

Purpose of PCR & Gel Electrophoresis PCRAmplifying a section of DNAGel Electrophoresis Sort DNA by sizeRead the sequence of a DNA fragment

DNA ArraysMicroscopic spot containing identical single-stranded molecules of DNAs, usually oligonucleotides or complementary DNAsAnalyze patterns of gene expression. DNA microarrays often consist of glass slides with spots of attached DNA fragments. The DNA fragments act as probes for specific sequences in a sample.Animation . Animation

Stem Cells Important to biomedical researchers because they can be used to generate virtually any type of specialized cell in the human body. Extracted from the egg after it has divided for 5 days. The egg at this stage of development is called a blastocyst. The extraction process destroys the embryoRaises ethical concerns. Hope that one day stem cells can be used to serve as replacement cells to treat heart disease, Alzheimer's, cancer, and other diseases.Animation

Pluripotent

Refers to a stem cell that has the potential to differentiate into any of the three germ layers: Endoderm (interior stomach lining, gastrointestinal tract, the lungs)mesoderm (muscle, bone, blood)Ectoderm (epidermal tissues and nervous system).

Pluripotent stem cells can give rise to any fetal or adult cell type. However, alone they cannot develop into a fetal or adult animal because they lack the potential to contribute to extraembryonic tissue, such as the placenta.

Totipotent

One of the most important stem cells types because they have the potential to develop into any cell found in the human body. In human development, the egg cell in a woman and the sperm cell from a man fuse together to form a single cell called the zygote. The zygote divides numerous times and forms cells that are the precursors to the trillions of cells that will eventually constitute the human body.

Practical applications of DNA Technology Gene therapy DNA transformation Animation AnimationGMOGenomic library

DNA library is a collection of cloned DNA fragments. There are two types of DNA library: The genomic library contains DNA fragments representing the entire genome of an organism. The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell. The advantage of cDNA library is that it contains only the coding region of a genomecDNA libraries can be used to clone cDNA for a known gene to discover the sequence of the mRNA it encodes

Use of complementary DNA (cDNA)Doublestranded DNA version of an mRNA molecule. In eukaryotes, an mRNA is a more useful predictor of a polypeptide sequence than is a genomic sequence, because the introns have been spliced out. Researchers prefer to use cDNA rather than mRNA itself because RNAs are inherently less stable than DNA and techniques for routinely amplifying and purifying individual RNA molecules do not exist. Animation

cDNA