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Recombinant DNA Technology. Deqiao Sheng PhD Dept. Of Biochemistry and Molecular Biology. Reference Books. Hortons’ Principles of Biochemistry 4th edition Molecular Biology , Robert Weaver 2nd edition Harpers’ Biochemistry 26th edition Styers’ Biochemistry.

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Recombinant DNA Technology

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    1. Recombinant DNATechnology Deqiao Sheng PhD Dept. Of Biochemistry and Molecular Biology

    2. Reference Books • Hortons’ Principles of Biochemistry 4th edition • Molecular Biology, Robert Weaver 2nd edition • Harpers’ Biochemistry 26th edition • Styers’ Biochemistry

    3. Recombinant DNA and Gene Cloning • Recombinant DNA (rDNA) is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together through the process of gene splicing. • Recombinant DNA technology is a technology which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future.

    4. Chinese Dragon

    5. The dragon is a mythical creature that can fly and walk. Dragon can change its form and has divine powers to summon wind and rain. • The dragons are said to be made up of many different types of animals of the Earth.

    6. Dragon is an imagination creature, which has deer's antlers, camel's head, hare's eye, snake's neck, carp's scales, eagle's claws, tiger's paws; and ox's ears. The Nine parts of A Chinese Dragon 

    7. Recombinant DNA:Cloning and Creation of Chimeric Genes

    8. Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists named Boyer and Cohen in 1973.

    9. Stanley N. Cohen , who received the Nobel Prize in Medicine in 1986 for his work on discoveries of growth factors. Stanley N. Cohen (1935–) (top) and Herbert Boyer (1936–) (bottom), who constructed the first recombinant DNA using bacterial DNA and plasmids.

    10. What is Recombinant DNA Technology? • Recombinant DNA technology is a technology which allows DNA to be produced via artificial means. • The procedure has been used to change DNA in living organisms and may have even more practical uses in the future. • It is an area of medical science that is just beginning to be researched in a concerted effort.

    11. Recombinant DNA technology works by taking DNA from two different sources and combining that DNA into a single molecule. That alone, however, will not do much. • Recombinant DNA technology only becomes useful when that artificially-created DNA is reproduced. This is known as DNA cloning.

    12. Brief Introduction

    13. Recombinant DNA Technology • The basic concepts for recombinant DNA technology • The basic procedures of recombinant DNA technology • Application of recombinant DNA technology

    14. The basic concepts for recombinant DNA technology

    15. In the early 1970s, technologies for the laboratory manipulation of nucleic acids emerged. In turn, these technologies led to the construction of DNA molecules composed of nucleotide sequences taken from different sources. The products of these innovations, recombinant DNA molecules, opened exciting new avenues of investigation in molecular biology and genetics, and a new field was born— recombinant DNA technology.

    16. Concept of Recombinant DNA • Recombinant DNA is a molecule that combines DNA from two sources . Also known as gene cloning. • Creates a new combination of genetic material • Human gene for insulin was placed in bacteria • The bacteria are recombinant organisms and produce insulin in large quantities for diabetics • Genetically engineered drug in 1986 • Genetically modified organisms are possible because of the universal nature of the genetic code!

    17. Genetic engineering is the application of this technology to the manipulation of genes. These advances were made possible by methods for amplification of any particular DNA segment( how? ), regardless of source, within bacterial host cells. Or, in the language of recombinant DNA technology, the cloning of virtually any DNA sequence became feasible.

    18. Recombinant technology begins with the isolation of a gene of interest (target gene). The target gene is then inserted into the plasmid or phage (vector) to form replicon. • The replicon is then introduced into host cells to cloned and either express the protein or not. • The cloned replicon is referred to as recombinant DNA. The procedure is called recombinant DNA technology. Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells.

    19. Some other terms are also in common use to describe genetic engineering. • Gene manipulation • Recombinant DNA technology • Gene cloning (Molecular cloning) • Genetic modification

    20. Cloning——In classical biology, a clone is a population of identical organisms derived from a single parental organism. • For example, the members of a colony of bacterial cells that arise from a single cell on a petri plate are clones. Molecular biology has borrowed the term to mean a collection of molecules or cells all identical to an original molecule or cell.

    21. Recombinant DNA technology——A series of procedures used to join together (recombine) DNA segments. A recombinant DNA molecule is constructed (recombined) from segments from 2 or more different DNA molecules. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate there, autonomously (on its own) or after it has become integrated into a chromosome.

    22. How recombinant technology works • These steps include isolating of the target gene and the vector, specific cuttingof DNA at defined sites, joining or splicing of DNA fragments, transforming of replicon to host cell,cloning, selecting of the positive cells containing recombinant DNA, and either express or not in the end.

    23. Six steps ofRecombinant DNA • Isolating (vector and target gene) • Cutting (Cleavage) • Joining (Ligation) • Transforming • Cloning • Selecting (Screening)

    24. Recombinant DNA Technology • The basic concepts for recombinant DNA technology • The basic procedures of recombinant DNA technology • Application of recombinant DNA technology

    25. The basic procedures of recombinant DNA technology

    26. DNA molecules that are constructed with DNA from different sources are called recombinant DNA molecules. • Recombinant DNA molecules are created in nature more often than in the laboratory; • for example, every time a bacteria phage or eukaryotic virus infects its host cell and integrates its DNA into the host genome, a recombinant is created. • Occasionally, these viruses pick up a fragment of host DNA when they excise from their host’s genome; these naturally occurring recombinant DNA molecules have been used to study some genes.

    27. Six basic steps are common to most recombinant DNA experiments • Isolation and purification of DNA. Both vector and target DNA molecules can be prepared by a variety of routine methods, which are not discussed here. In some cases, the target DNA is synthesized in vitro.

    28. 2. Cleavage of DNA at particular sequences. As we will see, cleaving DNA to generate fragments of defined length, or with specific endpoints, is crucial to recombinant DNA technology. The DNA fragment of interest is called insert DNA. In the laboratory, DNA is usually cleaved by treating it with commercially produced nucleases and restriction endonucleases.

    29. 3. Ligationof DNA fragments. A recombinant DNA molecule is usually formed by cleaving the DNA of interest to yield insert DNA and then ligating the insert DNA to vector DNA (recombinant DNA or chimeric DNA). DNA fragments are typically joined using DNA ligase (also commercially produced). • T4 DNA Ligase

    30. 4. Introduction of recombinant DNA into compatible host cells. In order to be propagated, the recombinant DNA molecule (insert DNA joined to vector DNA) must be introduced into a compatible host cell where it can replicate. The direct uptake of foreign DNA by a host cell is called genetic transformation (or transformation). Recombinant DNA can also be packaged into virus particles and transferred to host cells by transfection.

    31. 5. Replication and expression of recombinant DNA in host cells. Cloning vectors allow insert DNA to be replicated and, in some cases, expressed in a host cell. The ability to clone and express DNA efficiently depends on the choice of appropriate vectors and hosts.

    32. 6. Identification of host cells that contain recombinant DNA of interest. Vectors usually contain easily scored genetic markers, or genes, that allow the selection of host cells that have taken up foreign DNA. The identification of a particular DNA fragment usually involves an additional step—screening a large number of recombinant DNA clones. This is almost always the most difficult step.

    33. DNA cloning in a plasmid vector permits amplification of a DNA fragment.

    34. First step:Isolating DNA • Vector • Target gene

    35. How to get a target genes? • Genomic DNA • Artificial synthesis • PCR amplification • RT-PCR

    36. Polymerase chain reaction(PCR) • A technique called the polymerase chain reaction (PCR) has revolutionized recombinant DNA technology. It can amplify DNA from as little material as a single cell and from very old tissue such as that isolated from Egyptian mummies, a frozen mammoth, and insects trapped in ancient amber.

    37. method is used to amplify DNA sequences • The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube InitialDNAsegment Number of DNA molecules

    38. PCR primers

    39. RT-PCR • Reverse transcription polymerase chain reaction (RT-PCR) is a variant of polymerase chain reaction (PCR. • In RT-PCR, however, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional. • Template:RNA • Products: cDNA

    40. Vectors- Cloning Vehicles • Cloning vectors can be plasmids, bacteriophage, viruses, or even small artificial chromosomes. Most vectors contain sequences that allow them to be replicated autonomously within a compatible host cell, whereas a minority carry sequences that facilitate integration into the host genome.

    41. All cloning vectors have in common at least one unique cloning site, a sequence that can be cut by a restriction endonuclease to allow site-specific insertion of foreign DNA. The most useful vectors have several restriction sites grouped together in a multiple cloning site (MCS) called a polylinker.

    42. Types of vector • Plasmid Vectors • Bacteriophage Vectors • Virus vectors • Shuttle Vectors--can replicate in either prokaryotic or eukaryotic cells. • Yeast Artificial Chromosomes as Vectors

    43. Plasmid Vectors • Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s chromosomal DNA. • These extra chromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g., yeast), exist in a parasitic or symbiotic relationship with their host cell.

    44. Plasmid

    45. Plasmids can replicate autonomously within a host, and they frequently carry genes conferring resistance to antibiotics such as tetracycline, ampicillin, or kanamycin. The expression of these marker genes can be used to distinguish between host cells that carry the vectors and those that do not

    46. pBR322 • pBR322 was one of the first versatile plasmid vectors developed; it is the ancestor of many of the common plasmid vectors used in biochemistry laboratories. • pBR322 contains an origin of replication (ori) and a gene (rop) that helps regulate the number of copies of plasmid DNA in the cell. There are two marker genes: confers resistance to ampicillin, and confers resistance to tetracycline. pBR322 contains a number of unique restriction sites that are useful for constructing recombinant DNA.

    47. pBR322 • Origin of replication • Selectable marker • unique restriction sites

    48. Enzymes Restriction Enzymes and DNA Ligases Allow Insertion of DNA Fragments into Cloning Vectors • Restriction endonuclease, RE • DNA ligase • Reverse transcriptase • DNA polymerase, DNA pol • Nuclease • Terminal transferase

    49. Restriction enzymes cleave DNA • The same sequence of bases is found on both DNA strands, but in opposite orders. GAATTC CTTAAG • This arrangement is called a palindrome. Palindromes are words or sentences that read the same forward and backward. • form sticky ends: single stranded ends that have a tendency to join with each other ( the key to recombinant DNA)