Molecular Genetics Recombinant Dna And Genomic Technology

By: Jyoti Gurung Alisha Gurung Diwakar Chaudhary Nishma Bajracharya Manish Karmacharya

Molecular genetics is the field

of biology which studies the structure and function of genes at a molecular level. The field studies how the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology.

All DNA is made up of a base consisting of

sugar, phosphate and one nitrogen base. There are four nitrogen bases, adenine (A), thymine (T), guanine (G), and cytosine (C). The nitrogen bases are found in pairs, with A & T and G & C paired together and are complementary. The sequence of the nitrogen bases can be arranged in an infinite ways, and their structure is known as the famous "double helix“. The sugar used in DNA is deoxyribose. The four nitrogen bases are the same for all organisms. The sequence and number of bases is what creates

Double helical model of DNA

Recombinant

DNA is a form of artificial DNA that is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences that would not normally occur together. In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organismal genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose,

Restriction

enzymes: Certain endonucleases – the enzymes that cut DNA at specific DNA sequences within the molecule, are a key tool in recombinant DNA research. These are called restriction enzymes. Vectors: A vector is a DNA molecule into which foreign DNA may be inserted which can then replicate in an appropriate cell.

Blunt-ended DNA : Two strands of

a DNA duplex having ends that are flush with each other. Sticky –ended DNA: Complementary single strands of DNA that protrude from opposite ends of a DNA duplex or from the ends of different duplex molecules. Plasmid: A small, extrachromosomal , circular molecule of DNA that

EnzymesA Alkaline phosphate DNA ligase DNA polymerase I Dnase I

Exonuclease III

Reverse transcriptase

Reaction

Primary Use

Dephosphorylates 5’ Removal of 5’ –PO4 ends of RNA and groups prior to kinetic DNA. labeling prevent self – Catalyzes bonds Joining oftoDNA molecules. ligation. between DNA molecules. Synthesizes double- Synthesis of doublestranded DNA from stranded cDNA; nick single stranded DNA. translation; generation of blunt ends from sticky Under appropriate Nick translation; mapping conditions, produces ends. of hypersensitive sites; single-stranded nicks in DNA. Removes

mapping protein-DNA interactions. DNA sequencing;

nucleotides from 3’ ends of DNA.

mapping of hypersensitive sites; mapping Synthesisprotein-DNA of cDNA from interactions. mRNA; RNA (5’end)

Synthesizes DNA from RNA template.

mapping studies.

 It can supply large amounts of materials that

could not be obtained by conventional purification methods (eg., interferon, tissue plasminogen activating factor)  It can provide human material (insulin, growth hormone)  Proteins for vaccines(example: hep-B) and for diagnostic testing.(eg. AIDS tests) can be obtained.  It is used for the production of recombinant pharmaceuticals and in forensic medicine.  It is also used to engineer plants that are more resistant to drought or temperature extremes, more efficient at fixing nitrogen, or that produce seeds containing the complete complement of the

The classic example of the use of

recombinant DNA technology is sickle cell disease which is caused by mutation of single base out of 3x109 in the genome, a T-A DNA substitution, which in turn results in an A-U change in the mRNA corresponding to the sixth codon of the ß globin gene. The altered codons specifies a different amino acid i.e.valine rather than glutamic acid,

Gene therapy is being studied as a

possible treatment for sickle cell anaemia. Researchers are looking to see whether a normal gene can be planted in the bone marrow of a person with sickle cell anaemia, and thus cause the body to produce normal red blood cells. Researchers also are studying the possibility of treatment to “turn off” the sickle cell gene or “turn on” a gene that makes

1. Isolating Gene The gene for producing HUMAN insulin protein is isolated. The gene is part of the DNA in a human chromosome. The gene can be isolated and then copied so that many insulin genes are available to work with. 2. Preparing target DNA First, a circular piece of DNA called a plasmid is removed from a bacterial cell. Special proteins are used to cut the plasmid ring open.

4. Insert ing Plasmid back into cell The bacterial DNA now contains the human insulin gene and is inserted into a bacteria. Scientists use very small needle syringes to move the recombined plasmid through the bacterial cell membrane. 5. Plasmid multiply Many plasmids with the insulin gene are inserted into many bacterial cells. The cells need nutrients in order to grow, divide, and live. While they live,

6. Target Cells Reproduce Human insulin protein molecules produced by bacteria are gathered and purified. The process of purifying and producing cow and pig insulin has been greatly reduced or eliminated. 7. Cells Produce Proteins Millions of people with diabetes now take human insulin produced by bacteria or yeast (biosynthetic insulin) that is

CLONING Cloning is the isolation and growth of a single, genetically uniform cell and its offspring. Recombinant DNA cloning is the isolation of a cell line which contains (and replicates) a single, unique recombinant DNA from a pool (LIBRARY) of many different recombinant DNA’s.

The first human clone, a healthy 7-pound

girl named Eve, was born the 26th of December 2002, according to Clonaid, a private company linked to the Raelian religious sect.

A

transgenic animal or Genetically modified organism is an organism whose genetic material has been altered using genetic engineering techniques i.e. DNA recombinant technology. Scientists have produced a number of transgenic creatures they hope will bring major benefits to mankind. For example, human genes have been put into pigs to allow the pigs to produce human insulin, a substance needed to control diabetes, one of the fastest-growing diseases in affluent countries.

Sheep are being altered to generate a protein that may fight the lung disease emphysema. Designer dairy products are also on the drawing boards. Geneticists hope to end up with a breed of cow that produces, for example, only low-fat milk. Finally, researchers are optimistic that they will be able to turn animals, principally pigs, into sources of organs for human transplants. To

Transgenic animal

Ordinary zebra fish Gold Fish are a type of zebrafish with recombinant DNA. Genes for fluorescent proteins have been inserted into their genome to produce their fluorescent colors

 The human genome is composed of about 3 billion

base pairs of As and Ts, Gs and Cs. In 1990, an international consortium of scientists set out to create a map that would show exactly where on our twenty-three pairs of chromosomes every one of those base pairs is located. The effort, called the Human Genome Project, is the most extensive scientific enterprise ever undertaken.  Genetic mapping is the first step in isolating a gene. There are two basic approaches to this complex endeavor. The first is called linkage mapping, and its goal is to show where on each chromosome each gene is located relative to other genes. The method is to compare the genetic makeup of members of a family or closely related group of people who have a history of a

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