"More Tech Stuff"

Plasmid method | Genes: from Source to Send | Gene Selection | Hybridization |  Electrophoresis | Sanger Method | PCR | Blotting | RFLP analysis | In vitro mutagenesis | Human Genome Project | Commercial genetics

Bacteria and Plasmid Method

1. isolate plasmid and the DNA containing the gene of interest

2. plasmid and gene-o-interest digested with some enzyme

3. gene mixed with the clipped plasmid

4. ligase joins fragments permanently

5. altered plasmid introduced into cell

6. e.g. if lac Z interrupted than no beta-galactosidase thus a white color because X-gal isn't cleaved

7. e.g. if lac Z not broken than those bacterial colonies will be blue signifying the presence of the cleaved product

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Sources of Genes

A genomic library is a set of thousands of DNA segments from a genome, each carried by a plasmid or phage

• Shotgun Method is the random sampling of a genome.  It is called "shotgun" because no gene is targeted for cloning

• How to make cDNA (complementary) that doesn't have introns so bacteria can better express these genes

  1. transcription in the cell
  2. hnRNA processing (see spliceosomes) converted to mRNA
  3. mRNA isolated from teh cell and reverse transcriptase is added
  4. cDNA strand is synthesized and mRNA degraded
  5. 2nd DNA strand synthesized using DNA polymerase

• cDNA has the potential to be transcribed and translated by bacteria BUT there are no control sequences for transcript and translation normally associated with this gene (therefore in-identical)

• cDNA production also yields inaccurate libraries because mRNA cannot be isolated from all the other mRNA in the cell

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Inserting the DNA

The method depends on the vector and the type of host cell.

1. Electroporation: a brief electrical pulse applied to a solution causes temporary membraneous holes (often pores) to form through which DNA can enter
2. Direct injection of DNA into a single euk. cell by microscopically small needles
3. DNA gun: DNA coated microscopically tiny metallic particles are fired into a colony of cells

• most common is bacterial transformation
• if a phage is used rDNA is packaged into protein coat and injected into bacteria
• yeast can be induced to uptake plasmids and even linear DNA strands (as can other eukaryote cells)

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Selecting a Gene of Interest

If the clones in the cDNA library actually translate the gene(s) into protein(s) then it is possible to screen all clones for the presence of that/those protein(s)

• Detecting the protein:  based on activity (if an enzyme) or structure using antibodies to attach to it
  however most often screening techniques rely on detecting the gene, not its product
• Screening the gene directly:  depends on base-pairing between gene and complementary on another nucleic acid molecule (called hybridization)

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Hybridization

• the complementary molecule is called the probe and is either DNA or RNA
• how probes can be synthesized
  1. they can be sorted based on size and shape through the use of multiple lasers and high-tech computer software, then cloned by PCR (Polymerase Chain Reaction, see below)
  2. if the amino acid (or direct) sequence is known, probes can be chemically synthesized
• hybridization can provide info on evolutionary relationships between the target and homologous ones in the same or other species
• it also allows scientists to learn about the natural form of the target in relationship to the cDNA
• the use of a probe to map a gene or chromosome is called in situ hybridization.  Fluorescent probes are called "paints" and the use of chromosome-specific paints is therefore called chromosome painting.
  1. probe base-pairs to metaphase spreads on a glass slide
  2. autoradiograph and chromosome staining or chromsome staining and fluorescence antibody detection reveal which chromsome and the area on that chromosome to which the probe bonded.

Spectral Karyotyping (SKY).  In this procedure all human chromosomes are painted onto the genome to provide a direct and instantaneous analysis of that genomes condition.  SKY is currently used in the study of tumors and evolutionary cytogenetics - the study of the differences in the organization of the human genome in other species.

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Gel Electrophoresis separates macromolecules (DNA, RNA, protein) on the basis of charge, size, and other physical properties

Sanger Method (DNA sequencing and synthesis) is a way of tagging DNA synthesis with ddATP, ddGTP, ddTTP, or ddCTP.  It produces the sequence of the nucleotides in the sample.

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Polymerase Chain Reaction:  a general protocol to quick in vitro DNA amplification

1. a sequence is targeted (note:  isolation is not necessary, nor is purification; could be anything!)
2. DNA polymerase is added to initiate DNA synthesis, a nucleotide supply (sometimes labeled for fluorescence microscopy), buffer, and primers that are chemically synthesized complementary portions of the target DNA
3. heat briefly
4. cool to allow primers to hybridize to portions of the target DNA
5. DNA polymerase extends primer
6. cycle repeated @ approximately 5 min. per cycle

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Southern Blotting

1. restriction fragment preparation (often consists of entire genome)
2. electrophoresis
3. blotting:  single strands are transferred to nylon membranes or special paper by capillary action from the gel...note: when blotting uses capillary action it is called Southern Blotting.
4. hybridization:  single-stranded radioactive DNA probe complementary to the gene of interest is added and attaches by base-pairing to similar restriction fragments in the gel
5. rinse off excess probe
6. autoradiography:  radioactivity exposes teh film to form an image that corresponds to teh DNA fragments that base-paired with the probe
7. Northern blotting - the mainstay of gene expression research - works with mRNA.

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RFLP Analysis

• restriction fragment length polymorphisms are dfferences in DNA sequence on homologous chromosomes that result in differing restriction sites and thus differing restriction fragment patterns
• RFLPs are detected usually through Southern Blotting
• Since different sequences would result in different restriction sites for the same restriction enzyme(s), different electrophoresis results are expected

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Note:  Researchers often clone and identify genes without knowing its product.

In Vitro Mutagenesis:  one powerful approach

1. mutations often alter the function of the protein product.
2. when a mutated gene is reintroduced to the host cell, it is sometimes possible to determine the function of the missing normal protein by examining changes in the physiology or developmental pattern.

     A general protocol

1. plasmid denatured to 1st strand
2. mutagneic primer base-pairs
3. DNA polymerase elongates the primer
4. DNA ligase seals it
5. mutagenic vector invades bacteria
6. .50 mutant and .50 normal allows for a comparison to help identify the normal protein

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Human Genome Project

1. genetic mapping:  
   locate 3000 genes (identifiable loci called genetic markers) spaced evenly throughout the chromosomes.  It is possible because of so many RFLPs:  RFLP markers are genetic markers
2. physical mapping:
   cut each chromosome into identifiable fragments to determine their order by chromsome walking
   1. probe binds to known gene
   2. cut starting DNA with 2 different enzymes creating 2 libraries
   3. use probe 1 to screen library 2 for DNA fragments that overlap the known gene
   4. isolate DNA from library 2 clone tagged by the probe and make a new probe from the right end of the tagged clone
   5. probe 2 screens library 1 for overlapping segments
   6. repeat steps 4 and 5 with alternating libraries to "walk" down the original DNA
3. genome sequencing:
   determine exact nucleotide order
4. analyze genomes of other species to learn about them and develop new strategies and techniques for mapping the entire human genome

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Commercial Genetics:  DNA+Industry; Gene Therapy; Societal, Environmental, and Agricultural technologies

DNA and Industry

One objective of many:  Diagnose genetic diseases before birth to prevent genetic disorders.

Gene Therapy

Objective:  replace an allele by genetic recombination.  this works well with single enzyme deficiency diseases.  how?  put in bone marrow or remove cells and infect those with retrovirus vectors.
Questions:  when is gene therapy most effective?
          how do we get the "antidote" to the problem area?
          how do we assure regulation of the new gene?
          ...and other social and ethical questions.

Vaccines:

• The Problem and The Solution:  1 virus can equate to many diseases; therefore through the study of one virus it is possible to develop many vaccines for many diseases simultaneously.
• notes:  most pharmaceutical products are hormones (e.g. insulin) and proteins (usually interferon anticancer molecules).
• examples
  1. HGH (human growth hormone):  treats hypopituitarism (hypo- less, pituitar- pituitary gland production).
  2. EPO (erythropoietin):  stimulates red blood cell production in bone marrow while originating from the kidney; treats anemia.
  3. TPA (tissue plasminogen activation):  a very expensive way to dissolve blood clots
• a few other methods of fighting detrimental genes
  1. antisense nucleic acid:  a single-stranded RNA or DNA molecule that binds to mRNA to block translation of virulent or cancerous genes
  2. surface receptor mimicking to decoy a virus' presence in the body

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DNA Fingerprinting and VNTRs

DNA fingerprinting is RFLP analysis by electrophoresis and autoradiography. A major part of fingerprinting is the Variable Number Tandom Repeat (VNTR).  VNTRs are microsatellite DNA sequences at several loci in the human genome whose number of repeats varies within human populations.  Sometimes coupled with PCR, variable number tandem repeats - in this situation - measures the size of satellite DNA within a restriction fragment.

Environment and DNA Technology

Recycle wastes and detoxification with microorganisms that can be genetically engineered.

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Agriculture and DNA Technology

• popular tools in this area are the additions of cellulase in agriculture and Bovine Growth Hormone in the cattle and dairy industries
• transgenic farms attempt to make their crop develop faster with desired traits
• transgenic organism production is easier in plants because often one plant cell can grow into a whole plant
• the "best" vector is the Ti (Tumor inducing) plasmid of Agrobacterium tumefaciens.  It inserts DNA into the host's to cause tumors; however, only dicots (trees with 2 seed leaves) are susceptible to this vector.  Instead, the DNA gun previously described and electroporation are used for monocots (trees with 1 seed leaf).

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Next:  "Evolution: Nomenclature."