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Cloning a gene of an organism with an nonsequenced genome

Cloning a gene of an organism with an nonsequenced genome



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What would be the best protocol to clone a gene about 5kb in size? The genome is not sequenced, but the gene itself very similar to orthologous genes of organisms with known sequences.


I assume that we are talking about eukaryotic genomes which are split into introns and exons. Depending on what you want to do with your cloned gene, you need different strategies. The method is based on the assumption that the gene sequence has only very little differences to the known sequences of other organisms.

If you want to express your gene from a vector, you need to find the right processed mRNA which is then processed into cDNA and then cloned. Here two strategies are possible: If you have a gene, which is strongly expressed, you can design a primer primer (based on the orthologous sequences) which is anchored in the poly-A tail and has a gene specific seuqence next to it. With this it should be possible to amplify your gene specifically and isolate it. If the gene is not strongly expressed (the resulting amount of cDNA would be very small, since there is no amplification involved), I would opt for making cDNA from total mRNA (use anchored polyA primers) and then amplify the sequence from the cDNA pool with two primers (again based on the orthologous sequences) to amplify the gene sequence. I would go for TA-cloning here, since you do not know, which restriction enzyme recognition sites are present on your gene.

When you need the complete sequence containing introns and exons, there are two possible strategies. First you can use primers to amplify the gene and subsequently clone it. Then it is possible (and this is probably what I would do) to use a set of primers which bind to the end of the gene (and also the reverse strand) to amplify the sequences at the end and then sequence these PCR products. This gives you the exact sequences at the 5' and 3' end of the gene and allows you to include possible regulatory sites, since you then can design exactly matching primers for this region. Again, I would go for TA cloning.

And finally you could start by sequencing the whole region based on homologous sequence primers first and then with primers designed on the newly analyzed sequences. This is the most exact way, but is also costly and takes some time (since you need to make new primers after each round of sequencing until you are done).


Molecular Cloning

Cloning allows for the creation of multiple copies of genes, expression of genes, and study of specific genes. To get the DNA fragment into a bacterial cell in a form that will be copied or expressed, the fragment is first inserted into a plasmid. A plasmid (also called a vector in this context) is a small circular DNA molecule that replicates independently of the chromosomal DNA in bacteria. In cloning, the plasmid molecules can be used to provide a “vehicle” in which to insert a desired DNA fragment. Modified plasmids are usually reintroduced into a bacterial host for replication. As the bacteria divide, they copy their own DNA (including the plasmids). The inserted DNA fragment is copied along with the rest of the bacterial DNA. In a bacterial cell, the fragment of DNA from the human genome (or another organism that is being studied) is referred to as foreign DNA to differentiate it from the DNA of the bacterium (the host DNA).

Figure 1 Plasmids occur naturally in bacteria, but can also be modified by scientists.

Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids have been highly engineered as vectors for molecular cloning and for the subsequent large-scale production of important molecules, such as insulin. A valuable characteristic of plasmid vectors is the ease with which a foreign DNA fragment can be introduced. These plasmid vectors contain many short DNA sequences that can be cut with different commonly available restriction enzymes. Restriction enzymes (also called restriction endonucleases) recognize specific DNA sequences and cut them in a predictable manner they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction enzymes make staggered cuts in the two strands of DNA, such that the cut ends have a 2- to 4-nucleotide single-stranded overhang. The sequence that is recognized by the restriction enzyme is a four- to eight-nucleotide sequence that is a palindrome. Like with a word palindrome, this means the sequence reads the same forward and backward. In most cases, the sequence reads the same forward on one strand and backward on the complementary strand. When a staggered cut is made in a sequence like this, the overhangs are complementary (Figure 2).

Figure 2 In this (a) six-nucleotide restriction enzyme recognition site, notice that the sequence of six nucleotides reads the same in the 5′ to 3′ direction on one strand as it does in the 5′ to 3′ direction on the complementary strand. This is known as a palindrome. (b) The restriction enzyme makes breaks in the DNA strands, and (c) the cut in the DNA results in “sticky ends”. Another piece of DNA cut on either end by the same restriction enzyme could attach to these sticky ends and be inserted into the gap made by this cut.

Because these overhangs are capable of coming back together by hydrogen bonding with complementary overhangs on a piece of DNA cut with the same restriction enzyme, these are called “sticky ends.” The process of forming hydrogen bonds between complementary sequences on single strands to form doublestranded DNA is called annealing. Addition of an enzyme called DNA ligase, which takes part in DNA replication in cells, permanently joins the DNA fragments when the sticky ends come together. In this way, any DNA fragment can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction enzyme (Figure 3).

Figure 3: This diagram shows the steps involved in molecular cloning.

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they contain new combinations of genetic material. Proteins that are produced from recombinant DNA molecules are called recombinant proteins. Not all recombinant plasmids are capable of expressing genes. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins.


What is Cloning

Cloning refers to the creation of similar populations of genetically identical individuals. Hence, the clone has the same genetic material as the parent. Cloning occurs naturally through asexual reproduction. Bacteria and plants, as well as some animal forms, undergo asexual reproduction, producing genetically similar offspring. Hence, the clone and the parent organism have exactly similar phenotypic characteristics. A clone of trees is shown in figure 1.

Figure 1: Clone

The two types of cloning techniques used in biotechnology are molecular cloning and reproductive cloning.

Molecular Cloning

In molecular cloning, multiple copies of a particular gene can be produced as a clone. It is used in the study of a particular gene or the expression of the gene. First, the desired DNA fragment is inserted into a plasmid, and the plasmid can be transformed into bacteria along with the inserted fragment. The replication of the plasmid inside bacteria produces a large number of identical copies or clones of the plasmid with the insert. These clones can be either isolated from the bacterial cells or expressed inside the bacteria to obtain the gene product. Insulin-like proteins are produced through molecular cloning in a large scale. The formation of a recombinant plasmid is shown in figure 2.

Figure 2: Recombinant Plasmid

Reproductive Cloning

Reproductive cloning is the method of creating an identical copy of an entire multicellular organism. Most higher organisms use sexual reproduction where the fusion of haploid gametes forms a new diploid individual. Here, the diploid genetic complement and the cytoplasm of the egg cell are the two requirements for the production of an embryo. This approach can be artificially-produced by removing the haploid nucleus of the egg cell and placing the diploid, somatic nucleus of a donor into the egg cell. The procedure of reproductive cloning is shown in figure 3.

Figure 3: Reproductive Cloning

Then the egg cell is stimulated to divide, forming a new organism that consists of the same genetic information as the donor. Dolly was the first cloned agricultural animal who was born in 1996 and since then, goats, bulls as well as horses have been cloned.


Cloning

Cloning is a technique scientists use to create exact genetic replicas of genes, cells, or animals.

Biology, Genetics, Health, Chemistry

Cloned Beagles

Two beagle puppies successfully cloned in Seoul, South Korea. These two dogs were cloned by a biopharmaceutical company that specializes in stem cell based therapeutics.

Cloning is a technique scientists use to make exact genetic copies of living things. Genes, cells, tissues, and even whole animals can all be cloned.

Some clones already exist in nature. Single-celled organisms like bacteria make exact copies of themselves each time they reproduce. In humans, identical twins are similar to clones. They share almost the exact same genes. Identical twins are created when a fertilized egg splits in two.

Scientists also make clones in the lab. They often clone genes in order to study and better understand them. To clone a gene, researchers take DNA from a living creature and insert it into a carrier like bacteria or yeast. Every time that carrier reproduces, a new copy of the gene is made.

Animals are cloned in one of two ways. The first is called embryo twinning. Scientists first split an embryo in half. Those two halves are then placed in a mother&rsquos uterus. Each part of the embryo develops into a unique animal, and the two animals share the same genes. The second method is called somatic cell nuclear transfer. Somatic cells are all the cells that make up an organism, but that are not sperm or egg cells. Sperm and egg cells contain only one set of chromosomes, and when they join during fertilization, the mother&rsquos chromosomes merge with the father&rsquos . Somatic cells, on the other hand, already contain two full sets of chromosomes. To make a clone, scientists transfer the DNA from an animal&rsquos somatic cell into an egg cell that has had its nucleus and DNA removed. The egg develops into an embryo that contains the same genes as the cell donor. Then the embryo is implanted into an adult female&rsquos uterus to grow.

In 1996, Scottish scientists cloned the first animal, a sheep they named Dolly. She was cloned using an udder cell taken from an adult sheep. Since then, scientists have cloned cows, cats, deer, horses, and rabbits. They still have not cloned a human, though. In part, this is because it is difficult to produce a viable clone. In each attempt, there can be genetic mistakes that prevent the clone from surviving. It took scientists 276 attempts to get Dolly right. There are also ethical concerns about cloning a human being.

Researchers can use clones in many ways. An embryo made by cloning can be turned into a stem cell factory. Stem cells are an early form of cells that can grow into many different types of cells and tissues. Scientists can turn them into nerve cells to fix a damaged spinal cord or insulin-making cells to treat diabetes.

The cloning of animals has been used in a number of different applications. Animals have been cloned to have gene mutations that help scientists study diseases that develop in the animals. Livestock like cows and pigs have been cloned to produce more milk or meat. Clones can even &ldquoresurrect&rdquo a beloved pet that has died. In 2001, a cat named CC was the first pet to be created through cloning. Cloning might one day bring back extinct species like the woolly mammoth or giant panda.

Two beagle puppies successfully cloned in Seoul, South Korea. These two dogs were cloned by a biopharmaceutical company that specializes in stem cell based therapeutics.


Early cloning experiments

Reproductive cloning was originally carried out by artificial “twinning,” or embryo splitting, which was first performed on a salamander embryo in the early 1900s by German embryologist Hans Spemann. Later, Spemann, who was awarded the Nobel Prize for Physiology or Medicine (1935) for his research on embryonic development, theorized about another cloning procedure known as nuclear transfer. This procedure was performed in 1952 by American scientists Robert W. Briggs and Thomas J. King, who used DNA from embryonic cells of the frog Rana pipiens to generate cloned tadpoles. In 1958 British biologist John Bertrand Gurdon successfully carried out nuclear transfer using DNA from adult intestinal cells of African clawed frogs (Xenopus laevis). Gurdon was awarded a share of the 2012 Nobel Prize in Physiology or Medicine for this breakthrough.

Advancements in the field of molecular biology led to the development of techniques that allowed scientists to manipulate cells and to detect chemical markers that signal changes within cells. With the advent of recombinant DNA technology in the 1970s, it became possible for scientists to create transgenic clones—clones with genomes containing pieces of DNA from other organisms. Beginning in the 1980s mammals such as sheep were cloned from early and partially differentiated embryonic cells. In 1996 British developmental biologist Ian Wilmut generated a cloned sheep, named Dolly, by means of nuclear transfer involving an enucleated embryo and a differentiated cell nucleus. This technique, which was later refined and became known as somatic cell nuclear transfer (SCNT), represented an extraordinary advance in the science of cloning, because it resulted in the creation of a genetically identical clone of an already grown sheep. It also indicated that it was possible for the DNA in differentiated somatic (body) cells to revert to an undifferentiated embryonic stage, thereby reestablishing pluripotency—the potential of an embryonic cell to grow into any one of the numerous different types of mature body cells that make up a complete organism. The realization that the DNA of somatic cells could be reprogrammed to a pluripotent state significantly impacted research into therapeutic cloning and the development of stem cell therapies.

Soon after the generation of Dolly, a number of other animals were cloned by SCNT, including pigs, goats, rats, mice, dogs, horses, and mules. Despite those successes, the birth of a viable SCNT primate clone would not come to fruition until 2018, and scientists used other cloning processes in the meantime. In 2001 a team of scientists cloned a rhesus monkey through a process called embryonic cell nuclear transfer, which is similar to SCNT except that it uses DNA from an undifferentiated embryo. In 2007 macaque monkey embryos were cloned by SCNT, but those clones lived only to the blastocyst stage of embryonic development. It was more than 10 years later, after improvements to SCNT had been made, that scientists announced the live birth of two clones of the crab-eating macaque (Macaca fascicularis), the first primate clones using the SCNT process. (SCNT has been carried out with very limited success in humans, in part because of problems with human egg cells resulting from the mother’s age and environmental factors.)


17.1 Biotechnology

In this section, you will explore the following questions:

  • What are examples of basic techniques used to manipulate genetic material (DNA and RNA)?
  • What is the difference between molecular and reproductive cloning?
  • What are examples of uses of biotechnology in medicine and agriculture?

Connection for AP ® Courses

Did you eat cereal for breakfast or tomatoes in your dinner salad? Do you know someone who has received gene therapy to treat a disease such as cancer? Should your school, health insurance provider, or employer have access to your genetic profile? Understanding how DNA works has allowed scientists to recombine DNA molecules, clone organisms, and produce mice that glow in the dark. We likely have eaten genetically modified foods and are familiar with how DNA analysis is used to solve crimes. Manipulation of DNA by humans has resulted in bacteria that can protect plants from insect pests and restore ecosystems. Biotechnologies also have been used to produce insulin, hormones, antibiotics, and medicine that dissolve blood clots. Comparative genomics yields new insights into relationships among species, and DNA sequences reveal our personal genetic make-up. However, manipulation of DNA comes with social and ethical responsibilities, raising questions about its appropriate uses.

Nucleic acids can be isolated from cells for analysis by lysing cell membranes and enzymatically destroying all other macromolecules. Fragmented or whole chromosomes can be separated on the basis of size (base pair length) by gel electrophoresis. Short sequences of DNA or RNA can be amplified using the polymerase chain reaction (PCR). Recombinant DNA technology can combine DNA from different sources using bacterial plasmids or viruses as vectors to carry foreign genes into host cells, resulting in genetically modified organisms (GMOs). Transgenic bacteria, agricultural plants such as corn and rice, and farm animals produce protein products such as hormones and vaccines that benefit humans. (It is important to remind ourselves that recombinant technology is possible because the genetic code is universal, and the processes of transcription and translation are fundamentally the same in all organisms.) Cloning produces genetically identical copies of DNA, cells, or even entire organisms (reproductive cloning). Genetic testing identifies disease-causing genes, and gene therapy can be used to treat or cure an inheritable disease. However, questions emerge from these technologies including the safety of GMOs and privacy issues.

Information presented and the examples highlighted in the section support concepts outlined in Big Idea 3 of the AP ® Biology Curriculum Framework. The Learning Objectives listed in the Curriculum Framework provide a transparent foundation for the AP ® Biology course, an inquiry-based laboratory experience, instructional activities, and AP ® exam questions. A Learning Objective merges required content with one or more of the seven Science Practices.

Big Idea 3 Living systems store, retrieve, transmit and respond to information essential to life processes.
Enduring Understanding 3.A Heritable information provides for continuity of life.
Essential Knowledge 3.A.1 DNA, and in some cases RNA, is the primary source of heritable information.
Science Practice 6.4 The student can make claims and predictions about natural phenomena based on scientific theories and models.
Learning Objective 3.5 The student can justify the claim that humans can manipulate heritable information by identifying an example of a commonly used technology.
Big Idea 3 Living systems store, retrieve, transmit and respond to information essential to life processes.
Enduring Understanding 3.C The processing of genetic information is imperfect and is a source of genetic variation.
Essential Knowledge 3.C.1 Changes in genotype can result in changes in phenotype.
Science Practice 7.2 The student can connect concepts in and across domain(s) to generalize or extrapolate in and/or across enduring understandings and/or big ideas.
Learning Objective 3.24 The student is able to predict how a change in genotype, when expressed as a phenotype, provides a variation that can be subject to natural selection.

The Science Practice Challenge Questions contain additional test questions for this section that will help you prepare for the AP exam. These questions address the following standards:
[APLO 3.13][APLO 3.23][APLO 3.28][APLO 3.24][APLO 1.11][APLO 3.5][APLO 4.2][APLO 4.8]

Biotechnology is the use of biological agents for technological advancement. Biotechnology was used for breeding livestock and crops long before the scientific basis of these techniques was understood. Since the discovery of the structure of DNA in 1953, the field of biotechnology has grown rapidly through both academic research and private companies. The primary applications of this technology are in medicine (production of vaccines and antibiotics) and agriculture (genetic modification of crops, such as to increase yields). Biotechnology also has many industrial applications, such as fermentation, the treatment of oil spills, and the production of biofuels.

Basic Techniques to Manipulate Genetic Material (DNA and RNA)

To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base) linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases. The two strands can be separated by exposure to high temperatures (DNA denaturation) and can be reannealed by cooling. The DNA can be replicated by the DNA polymerase enzyme. Unlike DNA, which is located in the nucleus of eukaryotic cells, RNA molecules leave the nucleus. The most common type of RNA that is analyzed is the messenger RNA (mRNA) because it represents the protein-coding genes that are actively expressed. However, RNA molecules present some other challenges to analysis, as they are often less stable than DNA.

DNA and RNA Extraction

To study or manipulate nucleic acids, the DNA or RNA must first be isolated or extracted from the cells. Various techniques are used to extract different types of DNA (Figure 17.2). Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired (such as degradation of unwanted molecules and separation from the DNA sample). Cells are broken using a lysis buffer (a solution which is mostly a detergent) lysis means “to split.” These enzymes break apart lipid molecules in the cell membranes and nuclear membranes. Macromolecules are inactivated using enzymes such as proteases that break down proteins, and ribonucleases (RNAses) that break down RNA. The DNA is then precipitated using alcohol. Human genomic DNA is usually visible as a gelatinous, white mass. The DNA samples can be stored frozen at –80°C for several years.

RNA analysis is performed to study gene expression patterns in cells. RNA is naturally very unstable because RNAses are commonly present in nature and very difficult to inactivate. Similar to DNA, RNA extraction involves the use of various buffers and enzymes to inactivate macromolecules and preserve the RNA.

Gel Electrophoresis

Because nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, they can be mobilized by an electric field. Gel electrophoresis is a technique used to separate molecules on the basis of size, using this charge. The nucleic acids can be separated as whole chromosomes or fragments. The nucleic acids are loaded into a slot near the negative electrode of a semisolid, porous gel matrix and pulled toward the positive electrode at the opposite end of the gel. Smaller molecules move through the pores in the gel faster than larger molecules this difference in the rate of migration separates the fragments on the basis of size. There are molecular weight standard samples that can be run alongside the molecules to provide a size comparison. Nucleic acids in a gel matrix can be observed using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the top of the gel (the negative electrode end) on the basis of their size (Figure 17.3). A mixture of genomic DNA fragments of varying sizes appear as a long smear, whereas uncut genomic DNA is usually too large to run through the gel and forms a single large band at the top of the gel.

Amplification of Nucleic Acid Fragments by Polymerase Chain Reaction

Although genomic DNA is visible to the naked eye when it is extracted in bulk, DNA analysis often requires focusing on one or more specific regions of the genome. Polymerase chain reaction (PCR) is a technique used to amplify specific regions of DNA for further analysis (Figure 17.4). PCR is used for many purposes in laboratories, such as the cloning of gene fragments to analyze genetic diseases, identification of contaminant foreign DNA in a sample, and the amplification of DNA for sequencing. More practical applications include the detection of genetic diseases.

DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR) . The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it.

LINK TO LEARNING

Deepen your understanding of the polymerase chain reaction by clicking through this interactive exercise.

  1. The process of PCR can isolate a particular piece of DNA for copying, which allows scientists to copy millions of strands of DNA in a short amount of time.
  2. The process of PCR can purify a particular piece of DNA, and very small amounts of DNA can be used for purification.
  3. The process of PCR separates and analyzes DNA and its fragments, which requires very little DNA.
  4. The process of PCR anneals DNA molecules to complementary DNA strands, which maintains the same amount of DNA.

Hybridization, Southern Blotting, and Northern Blotting

Nucleic acid samples, such as fragmented genomic DNA and RNA extracts, can be probed for the presence of certain sequences. Short DNA fragments called probes are designed and labeled with radioactive or fluorescent dyes to aid detection. Gel electrophoresis separates the nucleic acid fragments according to their size. The fragments in the gel are then transferred onto a nylon membrane in a procedure called blotting (Figure 17.5). The nucleic acid fragments that are bound to the surface of the membrane can then be probed with specific radioactively or fluorescently labeled probe sequences. When DNA is transferred to a nylon membrane, the technique is called Southern blotting , and when RNA is transferred to a nylon membrane, it is called northern blotting . Southern blots are used to detect the presence of certain DNA sequences in a given genome, and northern blots are used to detect gene expression.

Molecular Cloning

In general, the word “cloning” means the creation of a perfect replica however, in biology, the re-creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone an entire organism, researchers learned how to reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning.

Cloning small fragments of the genome allows for the manipulation and study of specific genes (and their protein products), or noncoding regions in isolation. A plasmid (also called a vector) is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, the plasmid molecules can be used to provide a "folder" in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, the fragment of DNA from the human genome (or the genome of another organism that is being studied) is referred to as foreign DNA , or a transgene, to differentiate it from the DNA of the bacterium, which is called the host DNA .

Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). Plasmids have been repurposed and engineered as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which a foreign DNA fragment can be introduced via the multiple cloning site (MCS) . The MCS is a short DNA sequence containing multiple sites that can be cut with different commonly available restriction endonucleases. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner they are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two strands of DNA, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, these are called “sticky ends.” Addition of an enzyme called DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, any DNA fragment generated by restriction endonuclease cleavage can be spliced between the two ends of a plasmid DNA that has been cut with the same restriction endonuclease (Figure 17.6).

Recombinant DNA Molecules

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. Proteins that are expressed from recombinant DNA molecules are called recombinant proteins . Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to be moved into a different vector (or host) that is better designed for gene expression. Plasmids may also be engineered to express proteins only when stimulated by certain environmental factors, so that scientists can control the expression of the recombinant proteins.


There are a number of negatives involved with reproductive cloning, and they are listed below. Some of the arguments are religious in nature.

  • Cloning children could foster an understanding that children can be designed and replicated to the parents’ wishes.
  • There would be a lack of uniqueness and violate convictions regarding human individuality and freedom. Clones could be seen as less than human compared with non-clones.
  • Children created by cloning would live in the shadow of the genetic donor, always having to live up to the expectations of a person they were created from. Imagine discovering that you are the clone of a child your parents lost, and you live your life always being compared to the dead child.
  • 95% of animal cloning has ended in failure due to genetic defects, and cloning is considered unsafe because of it.
  • Many believe that cloning is against God’s wishes because the clones would be created by man. Man would be playing God and create people that are unable to feel and empathize. There is also the belief that these clones would be created without a soul.

There are many more arguments against reproductive cloning, but this is a brief list of the main arguments. When thinking about cloning children designed and replicated to the parents’ wishes, consider the book My Sister’s Keeper in which Anna is the product of preimplantation genetic diagnosis and is born for the sole purpose of being a bone marrow plant for her older sister Kate.


Pros of Cloning

Since the first successful execution of the process in 1996, cloning has become a useful technique in the field of biotechnology. Through cloning, transgenic (organisms having genes of interest inserted in their genome) plants and animals are used to make clones from adults. The following are some of the pros of cloning.

1. It can help prevent the extinction of species

As many organisms in the planet approach endangerment and extinction, cloning appears to be a possible solution to restore populations. By utilizing the genetic material of already dead organisms, cloning can even contribute to expanding the diversity of gene pools.

  • Aside from that, the cloning of extinct animals and their successful revival will also allow scientists to fully study the species as living organisms, instead of just studying their remains.
  • Despite being considered as an artificial mode of reproduction, cloning is in fact very common in a natural setting. The oldest form of cloning, asexual reproduction, is exhibited by various organisms like insects, and microorganisms.

2. It can help increase food production

Another major advantage of cloning is that it can serve as a means to increase agricultural production, particularly livestock and fresh produce. By manipulating their biological processes, existing traits of interest are ensured with the absence of the genetic “lottery” and random arrangements in the genes during meiosis.

  • During cloning, the gene of interest, as well as the organism bearing that gene of interest, is replicated faster than those undergoing the natural process.
  • Because of this, the number of organisms produced at a given time also increases.

3. It can help couples who want to have children

Last but not the least is the use of cloning as a means to produce children for infertile and same-sex couples. Normally, couples would want to have children that are biologically theirs. Interestingly, the genetic manipulations to be done could now be targeted at giving the children the genetic traits of both of their parents.

  • Children can now be produced even without donor eggs or donor sperms. Same-sex couples would only need a “surrogate” parent to carry the clone until its birth.
  • Scientists who support this method believe that it would become justifiable for these couples to reproduce in this method, assuming the procedures could be done safely.


To insert a mammalian gene into a prokaryotic cell, two basic requirements must be met.

  • First, researchers must isolate the target mammalian gene from the genome as a whole.
  • Second, the researchers must find a way to ensure that the prokaryotic cell can express the mammalian gene correctly.

Creating and Isolating the Target Gene

The eukaryotic chromosome and selected bacterial plasmids to be used as a cloning vector are treated with a restriction endonuclease.

When the eukaryotic DNA fragments are combined with the broken plasmids, some of the plasmids recombine with eukaryotic DNA.

The plasmids are then returned to the host bacteria by simply culturing both in solution so that some of the bacteria will take up the plasmids.

However, many of the plasmids will not contain recombinant DNA of those that do, only a small portion will contain the target mammalian gene.

Therefore, the next step is to isolate bacterial colonies that contain the recombinant plasmids incorporating the target gene.

This step involves two stages of screening:

Stage 1: Identify the bacterial colonies that contain recombinant plasmids.

Only a portion of the bacteria will take up recombinant plasmids.

To identify those that do, researchers typically use plasmids carrying a particular genetic marker — that is, a trait that is easily identified.

Stage 2: Identify the bacteria containing the desired gene.

When the mammalian DNA is broken with an endonuclease, the result is likely to be hundreds or thousands of fragments. Of these, only a small fraction will contain the target gene.

As a result, another step is required to find those bacteria that contain a plasmid that includes the right gene.

Identifying these bacteria involves the use of a nucleic acid probe in a technique called nucleic acid hybridization.

If at least part of the nucleic acid sequence of the gene is known, this information can be used to construct a probe made of RNA or single-stranded DNA. The probe consists of a nucleic acid sequence complementary to the known gene sequence, along with a radioactive or fluorescent tag.

To employ the probe, DNA from each bacterial colony is first heated to separate its two strands and then mixed with a solution containing the nucleic acid probe.

The probe forms a base pair with its complementary sequence, making it possible for researchers to locate the tag to determine which bacterial colony contains the desired gene.

Once the colony has been identified, it can be cultured to produce the gene product.

Expressing Eukaryotic Genes in Prokaryote Vectors

First, the promoter sequence of a eukaryotic gene will not be recognized by the prokaryotic form of RNA polymerase.

To overcome this problem, researchers have developed a particular type of plasmid called an expression vector.

An expression vector is a plasmid that contains a prokaryotic promoter sequence just ahead of a restriction enzyme target site. Thus, when recombination occurs, the inserted DNA sequence will lie close to the bacterial promoter. The host cell then recognizes the promoter and transcribes the gene.

Second, a prokaryote does not contain the snRNA or spliceosomes necessary to remove introns from a eukaryotic pre-mRNA transcript.

This means that the mRNA transcript in a prokaryote will contain both coding and non-coding sequences, both of which will be translated by the cell.

The solution to this problem has been to develop artificial eukaryotic genes that do not contain introns.

Researchers first isolate finished mRNA from the cytoplasm of an eukaryotic cell.

The mRNA is then placed in a solution with an enzyme called reverse transcriptase, which creates a DNA strand complementary to the mRNA strand.

This DNA strand is then isolated and added to a solution containing DNA polymerase, which synthesizes another complementary DNA strand.

The result is a double-stranded molecule of DNA containing only the coding portions of the eukaryotic gene. This synthetic molecule is called copy DNA or cDNA.

Another solution to both of these problems is to use eukaryotic cells as cloning vectors.

Yeast cells are often used for this purpose, since they can be cultured easily. Some yeast cells also contain plasmids, so similar techniques can be used to insert recombinant DNA into the cloning vector.

Inserting DNA into Plant or Animal Vectors

In some cases, only plant or animal cells will contain all the enzymes necessary to correctly manufacture a desired protein. Such cells can be grown in cultures to serve as cloning vectors.

However, because these cells are more difficult to culture, it is harder to insert foreign DNA into them.

To get around this apparent barrier and place foreign genes into eukaryotic genomes, biologists have developed several methods.


Cloning a gene of an organism with an nonsequenced genome - Biology

Article Summary:

INTRODUCTION
Sir George Mendel, the Father of Genetics, in middle nineteen century speculated a set of law to describe the inheritance of biological characters. The basic assumption of this law describes that each character in an organism is controlled by a factor known as "Trait" presently known as" Gene". A milestone came in biological sciences when Avery, MacLeod & McCarty in 1944 ,explained the molecular nature of Gene and described that Deoxyribonucleotide ( DNA) is genetic material and gave rise to new branch of biological science known as" Molecular Biology". For better understanding of Gene, its function & for its manipulation, we now turn into new technology to advance the modern biological sciences. This technology is termed as Recombinant DNA technology or Genetic Engineering. This technique includes the process of Gene cloning and as we all know the word cloning means" identical copy".

Basis steps in Gene cloning
Gene cloning is the process in which identical copies of a particular gene are manufactured by utilizing molecular biology tools. Gene cloning method requires DNA vector which is small, circular DNA molecule present in bacteria and this DNA vector has natural talent to replicate with fidelity when gene or segment of DNA is inserted into these molecules with in host. Plasmid of bacterium, many plant and animal viruses acts as DNA vector. Few basic steps regarding Gene Cloning are as follows:

Step 1: Gene that is to be cloned is inserted into DNA vector using a class of manipulation enzymes known as Restriction endonuclease which cuts at specific site on DNA and DNA ligase which is required to join the DNA. This type of DNA is known as Recombinant
Step 2: The Recombinant DNA molecule get transformed into host which is usually bacterium.
Step 3: Within the host the Recombinant DNA molecule replicate producing clones of genes that is inserted into vector.
Step 4: When host cells divides, each cell contains numerous number of Recombinant DNA molecules. This is way by which inserted gene are amplified.

Conclusion:
Gene cloning starts the new age of modern Bio-Technology. Gene cloning is being used to address problem in all areas of agriculture production, Pharmacology, Environmental issues, Food and nutrition field etc. This method makes DNA sequencing more effective and rapid as the massive genome sequence project like Human Genome Sequence Project was completed in the year 2000.

Application of Gene cloning
This recombinant DNA technology revolution opens the wide range of methods to solve problems in medicinal field. This technique is used to detect the genes which are responsible for genetic disorder and this disorder can be corrected by "Gene therapy". Gene therapy is emerging as promising and potential cure for treatment of cancer.

Gene cloning technique is going to acquire vital position to produce many recombinant pharmaceuticals like human insulin, recombinant DNA vaccine, recombinant human growth hormone, recombinant factor 8, interferon and many recombinant human proteins. There have been attempts to use gene cloning to disturb the infection cycle of many human pathogen like AIDS virus (HIV).

As this technology touches every aspects of our life how can agricultural be left behind. This technology is also securing good position in improving crop yields, production of herbicides resistant plants, drought resistant plants, disease resistant plants.

Gene library of an organism is prepared by gene cloning. Gene library is a collection of clones sufficient in number to be likely to contain every single gene present in a particular organism.

Gene library can be prepared by taking total cell DNA. The total cell DNA is partially digested with restriction end nuclease. This digested DNA is cloned into suitable vector. These recombinant vectors are transformed into particular host where they replicate and produce clone of genes present in that organism. This gene library is used detect the function, structure of gene of our interest present in particular organism.

The medicinal biology and the agricultural field are not the only way in which gene cloning are being utilized, an emerging field known as Archeogenetics is also obtaining benefits from this technology. By examine DNA sequence in living being and fossil, archeologist has begun to understand the evolutionary origin of modern humans.

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