AYESHA MASRUR KHAN DECEMBER, 2013

MANIPULATING MOLECULES 2

Natural Selection Methods exist within nature that optimize the selection process -The entire genome (all the genes) of higher animals and plants are broken up into functional components known as exons and separated by regions called introns. -Special genes known as transposable elements serve to mix and match functional components of genes in an effort to maximize the likelihood of creating better genes and organisms. -There is some evidence that bacteria, one of the simplest organisms, had introns and exons in some past era, but lost them in favor of efficiency and other means of acquiring new DNA. Artificial Selection Man applies selective pressure -Selective breeding of certain animals/plants with desirable traits - Invitro fertilization: grow fertilized eggs with desired traits and discard the rest. 3

4 Genetic Manipulation Radiation & Mutagenic compounds Viruses (viral vector) Transposable elements Gene targeting Germline cells Somatic cells

 True genetic engineering is the creation of whole new genes and proteins, or even new organisms. By understanding the genetic code and random or specific proteins can be created quite readily, but creating new proteins precisely for a given purpose for example, to strongly catalyze a particular chemical reaction is difficult.  Research into the structure and folding of proteins may yield some answers. Mixing and matching of the components of known proteins and organisms may yet be mastered, but that is a large step beyond even the manipulation of known genes. 5

Principle steps involved in genetic manipulation Generation of DNA fragments using enzymes that recognize and cut DNA molecules at specific DNA sequences Splicing of these sequences to other DNA molecules that serve as Vectors. The vector can replicate autonomously and these facilitate the manipulation and identification of the newly created recombinant DNA molecule. Transfer of vector carrying inserted DNA segment into a cell, which it is to be replicated, a process called Transformation of the cell. Selection of those cells that carry the desired recombinant DNA and their replication as clones 6

1. Production of DNA Fragments Restriction enzymes 1. Restriction enzyme cuts sugar phosphate backbone 2. DNA fragment is added from another molecule cut by the same restriction enzyme. 3. Base pairing occurs 4. DNA ligase seals strands Recombinant DNA molecule  Enzymes that cut DNA wherever a specific nucleotide sequence occurs  These restriction sites are generally 4-6 base pairs long  The sites which differ for restriction endonucleases are palindromes, i.e. they exhibit two-fold rational symmetry  Enzymes that cut DNA wherever a specific nucleotide sequence occurs  These restriction sites are generally 4-6 base pairs long  The sites which differ for restriction endonucleases are palindromes, i.e. they exhibit two-fold rational symmetry Sticky end 7

1. Production of DNA Fragments-cont’d Sticky & Blunt ends Two types of Restriction endonucleases:  Type I restriction endonucleases catalyze both the methylation of the host DNA and the cleavage of unmethylated DNA.  Type II restriction endonucleases are simpler in that they only cleave double stranded DNA at or near a specific unmethylated recognition sequence; separate enzymes, specific methyltranferases known as “restriction methylases” catalyze methylation at the same recognition sequence to protect host DNA 8 Digestions of double stranded DNA generates : Ends with a short single-stranded sequences, producing 5’ or 3’ overhangs –sticky ends Ends that are without any single stranded sequences-blunt ends

1. Production of DNA Fragments-cont’d BIOFACT: The 1978 Nobel prize in Physiology or Medicine was shared by three scientists who discovered restriction endonucleases. BIOFACT: The 1978 Nobel prize in Physiology or Medicine was shared by three scientists who discovered restriction endonucleases. 9

2. Joining to a vector/carrier molecule Properties of a useful vector:  Vector should be fairly small DNA molecule to facilitate isolation and handling.  The vector DNA can be introduced into a host cell.  The vector contains a replication origin and so can replicate inside the host cell.  Cells containing the vector can usually be selected by a straightforward assay, most conventionally by allowing the growth of the host cell on a solid selective medium. Properties of a useful vector:  Vector should be fairly small DNA molecule to facilitate isolation and handling.  The vector DNA can be introduced into a host cell.  The vector contains a replication origin and so can replicate inside the host cell.  Cells containing the vector can usually be selected by a straightforward assay, most conventionally by allowing the growth of the host cell on a solid selective medium. Recombinant DNA can be introduced into host cell by a vector, which is used to physically carry DNA into a host cell. A host cell can bacterial, yeast, plant, insect or mammalian.  Common bacterial vectors include plasmids and phages. Plasmids are circular units of DNA that can be engineered to carry a gene of interest. A phage is a virus that injects DNA into bacteria. Viral vectors 10

3. Introduction of DNA into host cell Methods depend upon the type of host/vector system and range from very simple procedures to much more complicated and esoteric ones. 1. Transformation transformation refers to the uptake of plasmid DNA 2. Transfection transfection refers to the uptake of phage DNA Alternative DNA delivery methods  Microinjection: Use fine needle to inject DNA into the nucleus  Biolistic: shooting DNA into the cell by a specific tool called 'Gene Gun'. The DNA is used to coat microscopic tungsten particles known as microprojectile, which are accelerated on a macroprojectile by firing a gunpowder change. At one end of the gun, there is a small aperture that stops the macroprojectile but allows the microprojectile carry DNA into the cell and in some cases, stable transformation will occur.  Electroporation: By exposing the bacteria to a small pulse of electricity, it causes the plasmid to enter the cell. Using several hundred volts dispersed across a few millimeters will result in a merger of the two materials. 11

3. Introduction of DNA into host cell-cont’d 12 Sample delivery>> The helium pulse sweeps the DNA- or RNA-coated gold microcarriers from the inside wall of the sample cartridge. The microcarriers accelerate for maximum penetration as they move through the barrel, while the helium pulse diffuses outward. The spacer maintains the optimal target distance for in vivo applications and vents the helium gas away from the target to minimize cell surface impact.

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