Tuesday, 7 June 2011

Plasmid Isolation (Alkaline Lysis)

Bacterial plasmids, the non-genomic transferable DNA, can easily be purified from bacteria using numerous techniques.  The purification of DNA is important for genetic research as it provides a source of transferable DNA and allows researchers to isolate large amounts of recombinant DNA. One common technique for plasmid purification is the alkaline lysis method, which breaks open bacteria with an alkaline solution, proteins are removed by precipitation and the plasmid DNA is recovered with alcohol precipitation.
Students purify bacterial plasmids from a liquid culture using this alkaline lysis method. 

Protein biosynthesis

Protein biosynthesis (Synthesis) is the process in which cells build proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription which are then used for translation.

Protein biosynthesis, although very similar, differs between prokaryotes and eukaryotes.The events following biosynthesis include post-translational modification and protein folding. During and after synthesis, polypeptide chains often fold to assume, so called, native secondary and tertiary structures. This is known as protein folding. Amino acids are the monomers which are polymerized to produce proteins.

Amino acid synthesis is the set of biochemical processes (metabolic pathways) which build the amino acids from carbon sources like glucose. Not all amino acids may be synthesised by every organism, for example adult humans have to obtain 8 of the 20 amino acids from their diet. The amino acids are then loaded onto tRNA molecules for use in the process of translation.

RNA

Ribonucleic acid or RNA is a nucleic acid polymer consisting of nucleotide monomers that plays several important roles in the processes that translate genetic information from deoxyribonucleic acid (DNA) into protein products; RNA acts as a messenger between DNA and the protein synthesis complexes known as ribosomes, forms vital portions of ribosomes, and acts as an essential carrier molecule for amino acids to be used in protein synthesis. RNA is very similar to DNA, but differs in a few important structural details: RNA is single stranded, while DNA is double stranded.

Also, RNA nucleotides contain ribose sugars while DNA contains deoxyribose and RNA uses predominantly uracil instead of thymine present in DNA. RNA is transcribed from DNA by enzymes called RNA polymerases and further processed by other enzymes.

RNA serves as the template for translation of genes into proteins, transferring amino acids to the ribosome to form proteins, and also translating the transcript into proteins. RNA is a polymer with a ribose and phosphate backbone and four different bases: adenine, guanine, cytosine, and uracil.
The first three are the same as those found in DNA, but in RNA thymine is replaced by uracil as the base complementary to adenine. This base is also a pyrimidine and is very similar to thymine.

Uracil is energetically less expensive to produce than thymine, which may account for its use in RNA. In DNA, however, uracil is readily produced by chemical degradation of cytosine, so having thymine as the normal base makes detection and repair of such incipient mutations more efficient. Thus, uracil is appropriate for RNA, where quantity is important but lifespan is not, whereas thymine is appropriate for DNA where maintaining sequence with high fidelity is more critical.

Gene Testing

What is gene testing? How does it work?
Gene tests (also called DNA-based tests), the newest and most sophisticated of the techniques used to test for genetic disorders, involve direct examination of the DNA molecule itself. Other genetic tests include biochemical tests for such gene products as enzymes and other proteins and for microscopic examination of stained or fluorescent chromosomes. Genetic tests are used for several reasons, including:

  • carrier screening, which involves identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to be expressed

  • preimplantation genetic diagnosis (see the side bar, Screening Embryos for Disease)

  • prenatal diagnostic testing

  • newborn screening

  • presymptomatic testing for predicting adult-onset disorders such as Huntington's disease

  • presymptomatic testing for estimating the risk of developing adult-onset cancers and Alzheimer's disease

  • confirmational diagnosis of a symptomatic individual

  • forensic/identity testing 
In gene tests, scientists scan a patient's DNA sample for mutated sequences. A DNA sample can be obtained from any tissue, including blood. For some types of gene tests, researchers design short pieces of DNA called probes, whose sequences are complementary to the mutated sequences. These probes will seek their complement among the three billion base pairs of an individual's genome. If the mutated sequence is present in the patient's genome, the probe will bind to it and flag the mutation. Another type of DNA testing involves comparing the sequence of DNA bases in a patient's gene to a normal version of the gene. Cost of testing can range from hundreds to thousands of dollars, depending on the sizes of the genes and the numbers of mutations tested. 

Monday, 6 June 2011

Gene Therapy

Gene therapy is 'the use of genes as medicine'. It involves the transfer of a therapeutic or working gene copy into specific cells of an individual in order to repair a faulty gene copy. Thus it may be used to replace a faulty gene, or to introduce a new gene whose function is to cure or to favourably modify the clinical course of a condition.

The scope of this new approach to the treatment of a condition is broad, with potential in the treatment of many genetic conditions, some forms of cancer and certain viral infections such as AIDS.

Genes, which are carried on chromosomes, are the basic physical and functional units of heredity. Genes are specific sequences of bases that encode instructions on how to make proteins. Although genes get a lot of attention, it’s the proteins that perform most life functions and even make up the majority of cellular structures. When genes are altered so that the encoded proteins are unable to carry out their normal functions, genetic disorders can result.

Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:
  • A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.

  • An abnormal gene could be swapped for a normal gene through homologous recombination.

  • The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.

  • The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.
    How does gene therapy work?
    In most gene therapy studies, a "normal" gene is inserted into the genome to replace an "abnormal," disease-causing gene. A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of this capability and manipulate the virus genome to remove disease-causing genes and insert therapeutic genes.

    Target cells such as the patient's liver or lung cells are infected with the viral vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state. See a diagram depicting this process.

    Some of the different types of viruses used as gene therapy vectors:
    • Retroviruses - A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus.

    • Adenoviruses - A class of viruses with double-stranded DNA genomes that cause respiratory, intestinal, and eye infections in humans. The virus that causes the common cold is an adenovirus.

    • Adeno-associated viruses - A class of small, single-stranded DNA viruses that can insert their genetic material at a specific site on chromosome 19.

    • Herpes simplex viruses - A class of double-stranded DNA viruses that infect a particular cell type, neurons. Herpes simplex virus type 1 is a common human pathogen that causes cold sores.
    Besides virus-mediated gene-delivery systems, there are several nonviral options for gene delivery. The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with certain tissues and requires large amounts of DNA.

    Another nonviral approach involves the creation of an artificial lipid sphere with an aqueous core. This liposome, which carries the therapeutic DNA, is capable of passing the DNA through the target cell's membrane.

    Therapeutic DNA also can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell. This delivery system tends to be less effective than other options.

    Researchers also are experimenting with introducing a 47th (artificial human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 --not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic code, and scientists anticipate that, because of its construction and autonomy, the body's immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cell.

    DNA Viruses

    Most people encounter viruses at some point throughout their lifetime, which can leave them feeling miserable. We probably don't give a lot of thought to the molecular aspects of viruses and instead, just focus on getting rid of the painful symptoms when a virus strikes and compromises our health. If you stop for a minute and think about it, however, a lot is happening in our bodies when a virus invades. All of the symptoms you feel result from the collective effects of a virus as it inflicts your body's cells.

    How Do Viruses Work?
    A virus essentially inserts its own genetic material into a host cell and causes changes in the function of the host cell through the virus' genes. The host cell may lose many different abilities, such as control over its growth, its ability to divide and it may also show chromosomal abnormalities.

    Like humans, viruses are composed of genetic material. In the case of a DNA virus, this genetic material is DNA. A DNA virus uses a copying mechanism, which relies on an enzyme called DNA polymerase - an important enzyme in DNA replication. DNA viruses are grouped according to the Baltimore classification system and there are more than twenty virus families and nearly a hundred genera. The viruses themselves include an enormous range, from more basic ones to highly complex viruses.

    Different Kinds of DNA Viruses:
    There are numerous different groups of DNA viruses, with varying structures and effects. One group is herpes viruses, which are somewhat complex and have over one hundred genes. One of the herpes family of viruses is the Epstein Barr virus, which has been associated with a rare form of cancer. Epstein Barr virus is also the virus responsible for glandular fever - formally known as infectious mononucleosis, which is commonly seen in adolescents and college-aged adults. Pox viruses have several hundred genes and are effective at replicating once in a host. Papilloma viruses are those that cause warts and they are also associated with cancer. You have also likely heard of the Hepatitis B virus, which is actually a DNA tumour virus. There are many more DNA viruses but they all share a common link, which is that their genetic material is comprised of DNA.

    What are DNA Mutations?

    There are normal variations from one person to another. You can simply look at your friends and family to see the differences. These kinds of variations result from differences in our DNA. There are, however, differences that occur from abnormal variations in a gene and these can have detrimental consequences on a person such as disease.

    How do DNA Mutations Occur?
    Virtually every single person will have some sort of change to their DNA during their life. Changes can result from a multitude of mistakes, such as an error when DNA is replicated or through damage to DNA occurring from environmental or lifestyle factors. These include smoking, radiation and many others. Fortunately, your cells have special ways to handle these mistakes before they can cause damage. For some people, however, their body's repair systems can become overwhelmed if repeatedly exposed to a specific stimulus. For all of us as well, our DNA repair systems just do not operate nearly as successfully as we age. The end result for both of these scenarios is that changes in DNA will occur.

    A DNA mutation can also be inherited. A germline mutation is one that can result in a disease that is clustered within one family. Some mutations can be quite specific, such as those that occur following excessive exposure to sunlight, which can cause changes in skin cells. Still other mutations may occur in the area of DNA related to sperm and egg production, which is also considered a germline mutation and is inheritable. If your child were to inherit a germline mutation from you, each cell in your child's body would carry this faulty DNA.
    Different Types of Mutations:
    To understand the different types of mutations that can occur, it is important to know how a gene is constructed. Your DNA is full of genes, which are similar to words that make up a sentence. The four bases are known as adenine (A), thymine (T), guanine (G), and cytosine (C), each denoted by their first letter. Different sequences of these bases code for different proteins. If the sequence is modified, the entire meaning of the gene then changes and the instructions for producing the protein changes as well.

    Point mutations are those that involve a basic change in a single base for the sequence. If we removed just a single letter from in a word or sentence, this would be akin to a point mutation. In contrast, a frameshift mutation involves the addition or removal of nucleotides. At the same time, if you think about the fact that DNA reads in sequences of three bases or 'letters,' the addition or removal of one or more letters alters every word that follows as the letters are all shifted. Therefore, the entire meaning of the sentence is changed.

    Another type of mutation is a deletion mutation. Any mutation where DNA is ultimately missing a piece is called a deletion mutation. The mutation may be quite small and could involve deletion of only a single base or it could be larger and will impact numerous genes. A deletion mutation can even result in a frameshift mutation, where an entire 'word' is deleted. Conversely, a mutation that involves an additional piece of DNA is called an insertion mutation. In fact, these types of mutations can also result in frameshift mutations. Regardless of whether it is a deletion or insertion, a frameshift mutation usually translates into a protein that does not function properly.

    Other mutations include inversion and expression mutations. In the latter, a whole section of a person's DNA is actually reversed while in an expression mutation, it is not just the protein that may be changed but the location where it is made or the amount of the protein produced. So basically, if you had this mutation, your body could be making a protein in a skin cell, for example, when it should be making it in a nerve cell.

    Sunday, 5 June 2011

    What is a gene?

    The DNA double helix stores information in the form of a genetic code. Sections of DNA that contain complete messages are known as genes. They can be thought of as 'words' along the DNA 'sentences'.

    Genes are messages that provide the information for all cellular functions. They carry information that is passed on to future generations.

    An organism's genes determine:
    • the characteristics that are used to classify it into the plant or animal kingdom and into a specific family and species
    • how it uses food
    • how well it fights infection
    • at times, how it behaves.
    Each human cell (except red blood cells) contains between 25,000 and 42,000 genes. Genes control the production of proteins that make up most of your body.

    Genes code for proteins:
    Genes contain the coded formula needed by the cell to produce proteins. Proteins are the most common of the complex molecules in your body. Types of proteins include:
    • structural proteins, such as those which form hair, skin and muscle
    • messenger proteins, such as hormones, which travel around your body controlling such things as the sugar content of your blood
    • enzymes, which carry out most of the life processes inside your body, for example making haemoglobin for your red blood cells.

    What is DNA?

    Deoxyribonucleic acid (DNA) is a very important molecule found in all living cells. It contains information used in everyday metabolism and growth and influences most of our characteristics.

    DNA is often described as the blueprint of an organism. It enables various cells to develop and work together to form a fully functional body, and controls characteristics such as eye colour. How much DNA influences very complex features, such as intelligence, is not yet fully understood.

    The information that DNA contains is passed from one generation to the next. There is much debate over how much of what we are like is due to inheritance and defined by our DNA, and how much is defined by the influence of the environment. This is sometimes referred to as the 'nature/nurture' debate.

    Using gene technology, DNA can be modified or transferred from one organism to another. Genes are made up of short lengths of DNA and modern gene technology is able to make changes at the level of individual genes.

    Surprisingly, while the DNA molecule is very long, it is stunningly simple. DNA looks like an incredibly long twisted ladder. This shape is called a double helix.


    The sides of the ladder are a linked chain of alternating sugar and phosphate molecules. The rungs connect to the sugar molecules and are known as bases.

    There are four bases - adenine (A), thymine (T), guanine (G) and cytosine (C). Each rung is made up of two bases that link together. Because of their chemical nature, A will only link with T and G will only link with C.


    DNA from all living organisms is made of the same sugar and phosphate molecules and the same four bases. Whether DNA is in your cells, those of a cactus, of a worm or a bacterium, it is made of the same chemicals and has the same structure.

    The only difference is the order or the sequence of the bases in the DNA molecule. It is this sequence that is referred to as the genetic code, and why it is sometimes called the code of life.

    Saturday, 4 June 2011

    Genetic disorders

    The human race has a huge diversity in many features – skin colour, size, intellectual and athletic abilities, to name just a few. This variation has arisen largely due to:
    • changes in the DNA determining these features
    • changes to and interactions with the environment.
    Any change in our genes or DNA is called a mutation. All genetic variation has arisen from mutations. The different forms of a gene that arise through mutation are called alleles.

    While we most often associate mutations with genetic diseases, much of the variation has led to a highly complex body that works extremely well in our environment.

    Some mutations may change the gene so that it codes for a protein that works just as well, or maybe even better than, the protein coded for by the original gene.

    Unfortunately, however, some gene changes result in the production of a different protein that does not work as efficiently, or in the same manner, as the one usually coded for by that gene. In some cases, no functional protein is produced at all. In such cases, the mutation or gene change may cause a genetic condition or disease, such as cystic fibrosis or Huntington's disease.

    Approximately 3% of Australian babies may be born with a genetic condition where one or more genes that played an important role have been mutated.

    Genetic testing:

    Genetic tests look at a person's genetic material: genes or DNA. Such tests can compare the base sequences in sections of DNA, look at the results of a change or mutation that is present in the DNA, or examine the shape and structure of chromosomes.

    DNA is usually taken from a blood sample, but other body fluids or tissues may be used. The tests may look for predisposition to disease, or confirm a genetic mutation in an individual or family. As well as studying changes to chromosomes or genes, genetic testing also includes biochemical tests for certain proteins that indicate disease-causing gene variations.

    Carrier testing can determine if a couple is ‘carrying’ a particular gene mutation for an inherited disorder (such as cystic fibrosis) that they may pass on to their children.

    Predictive genetic testing focuses on tests that identify if someone will develop a disease before any symptoms appear. These tests can be useful for early detection, diagnosis, prognosis and treatment (if available).

    Genetic testing is also used to identify people with an increased risk or predisposition of developing a particular condition, such as certain cancers. This information may be useful in helping to prevent, treat or manage the disease, but it also raises many issues for our community.

    Newborn screening:

    In Australia, all newborn children are screened for several diseases, including:
    All or some of the symptoms of these disorders can be prevented, or their severity reduced, if the condition is diagnosed and treated early in life. Newborn screening programs use a blood sample obtained by a heelprick from babies about three days after birth.

    These blood samples are stored on a special pre-printed filter paper called a Guthrie card. Different chemicals and proteins are measured in this sample to determine whether the baby may have a particular disease. If these tests indicate that the baby may have a genetic condition such as cystic fibrosis, then their DNA may be tested to see if they carry a gene variant causing this.

    This provides information to the parents if they are hoping to have more children. Guthrie cards are stored by the laboratory doing the testing. Some people have expressed concerns about how the DNA in the blood of these cards may be used in the future.

    Friday, 3 June 2011

    Why do we do biotechnology?

    Biotechnology is used in a wide range of applications in food science, medicine, the environment and agriculture. Research is rapidly expanding the possibilities of where it will be used next.

    Any technology brings with it risks as well as benefits, and gene technology is no exception. These risks need to be carefully assessed before a genetically-modified (GM) plant, animal or microorganism is released.

    Government regulatory authorities assess the risks, which may include:
    • how readily the released organism could cross-breed with similar organisms in the environment
    • whether the modification gives the organism extra survival advantages
    • whether these advantages could upset a balanced ecosystem.

    What is Biotechnology?

    A general description of biotechnology is using living things to create products or to do tasks for human beings.

    Biotechnology is the practice of using plants, animals and micro-organisms such as bacteria, as well as biological processes - such as the ripening of fruit or the bacteria that break down compost - to some benefit.

    For example, biotechnology is used in in industry, medicine and agriculture to produce foods, medicines, test for diseases and remove waste.

    It can also be used to solve problems and conduct research. Over time, biotechnology has formed the basis of learning about people and diseases. Biotechnology has also underpinned the development of treatments.

    DNA contains all of the information required to grow and build a whole organism, from a bacterium to a plant, to a human. Studying the DNA of living organisms provides us with more information on how our body (or a plant or an animal) works and what happens when things go wrong.

    Biotechnology is the use of living organisms to create products or to do tasks for us. Researchers use DNA, genes, yeast, bacteria and cells to create things.