Applications of Biotechnology & Genomics
Biotechnology: “The use of biological systems to create goods & services" (Bio3950, Drs Marshall & Carr)
       Clinical :     Diagnosis & Treatment of human genetic diseases
       Forensic :  Data  Evidence in legal proceedings
                            Identification of unknown or questioned material
       Commercial : Production of transgenic products & organisms
                                 Improvement of production traits of agricultural species

Genomics: the study of complete gene sets (genomes) of organisms (Bio4251, Dr Rise)
        Proteomics: study of coordinate protein expression patterns across genomes (Bio4255, Dr Bykova)
                                "There is one genome, there are many proteomes."
        Bioinformatics:  computational analysis of genomic & proteomic data (Bio3951, Dr Peña-Castillo)
        Advanced Genetics: In-depth consideration of molecular & classical genetics (Bio4241, Dr Carr)
Restriction Fragment Length Polymorphism (RFLP) Analysis

  Restriction maps vary among  species &  individuals according to DNA sequence
       SNPs may alter restriction sites & produce Restriction Fragment Length Polymorphism (RFLPs)
       RFLPs are inherited in Mendelian co-dominant alleles

  Variation in the pattern of restriction digests identifies point mutations
   RFLPs may indicate point mutations (SNPs) directly
       Ex.: MstII RFLP test differentiates sickle-cell HbS versus HbA alleles [HOMEWORK]
   RFLPS can be genetic markers for specific alleles
       marker: restriction site shows presence / absence of genetically linked trait  [HOMEWORK]
            Markers do not cause the traits!
            Restriction fragment pattern indicates which trait alleles are present          

       Useful in combination with amniocentesis for in utero genetic diagnosis
            Cells, fluids, & DNA from at-risk fetus can be examined genetically

Allele-Specific Oligonucleotides (ASOs)

   Allelic differences are due to single SNPs
        ASOs are SNP-specific probes
        "Southern Blot" test without electrophoresis, probe "sticks" to only one allele:
            genetic screen is "positive or negative" result
            Ex.: 508-ASO Test for Cystic Fibrosis probes deletion mutation (OMIM219700)
       
    Pre-Natal Screening: the GATACCA model

       Ex.: Mr. & Ms. Mus seek genetic counselling ....
       Ex.: Pre-implantation Genetic Diagnosis (PGD)

See also Species-Specific Oligonucleotides (SSOs)
                Quantitative "Real-Time" PCR assays (RT-PCR)

DNA Fingerprinting
    Determination of an individual-specific genetic patterns
  
  Variable Number of Tandem Repeat (VNTR) loci

                Minisatellites - 15~100 nucleotide motifs repeated several times

                Microsatellites - di-, tri-, tetra-nucleotide motifs repeated 101s ~ 106s of times
                                              (CACACACACACA versus CACACACA) [IG1 21.02]
                 Origin by slipped-mismatch replication
         Copy number mutates rapidly, even between parents & children
             VNTR loci are highly polymorphic  [HOMEWORK] [IG1 21.03]
                  good marker for within-population studies [IG1 21.05]
                  [Animation of DNA Fingerprinting for forensics ]


        Forensics - use of DNA data as legal evidence [IG1 21.04]
             Positive dentification of individuals [HOMEWORK] with high probability [IG1 21.06] [IG1 19.12]

DNA "Chips"
       Oligonucleotide probes ("oligos") immobilized on a glass chip
              randomly-cut genomic DNA is tagged & used to "probe" the chip
              [click here for an animation of DNA microarrays]

      "Variant Detector Arrays (VDAs)" detect specific SNP alleles in genome                                                             

       "GeneChips" perform automated "Northern" analysis:
             specific mRNAs or cDNAs are detected in proteome
            "pseudo-colours" indicate levels of gene expression

       DNA "re-sequencing arrays" sequence of 30~500 Kbp in one experiment

Transgenic organisms

Genomics: the genetic study of entire genomes

    The Human Genome Project  (sponsored by HuGO)
               A statement of genetic ethics (Lee et al. 2008)
     3.2 x 109 basepair (3,200 Mbp) genome sequence completed [IG1 19.02]
                10-year project: "first draft" announced: March 2001 (Venter et al. 2001)
                     "Finished draft" of euchromatic sequence, October 2004
                     First personal genome sequence: James Watson, June 2007
                 Similar projects completed in Drosophila & Mouse
                    Genome projects underway in multiple species [Current list] [IG1 19.02]

   'Top Down' Strategy: Move from large-scale  fine-scale map
            Large-scale mapping of chromosomes  provides "scaffold"
            Fine-scale mapping of individual genes
            Sequence contigs   complete genome

  Insights: ~ 20,500 protein-coding genes in human genome [IG1 19 Table02]
              ~6,000 have unknown functions [IG1 19.03]
                  Expressed Sequence Tags (ESTs)
                       partial sequences of 5' ends of cDNAs reverse transcribed from mRNA [IG1 22.12]
                           cDNA   DNA sequence expressed as mRNA  presumptive "gene"
                           Sequences of ESTs with unknown functions from "non-model" species
                       compared with genes of known function from "genome-enabled" species

              only  ~ 7,000 novel genes in Homo wrt Drosophila [IG1 19.04]
               Comparison of Homo wrt Chimpanzee (Pan) genome: ~ 98.44% similar [see exercise below]
                   Differences? "silenced" human gene for Caspase-12 implicated in Alzheimers
  
             >60% of genome without ordinary genetic function [IG1 19.06]
                       repetitive DNA arranged tandem or dispersed repeats [IG1 19.09]
                       Alu family constitutes 11% of human genome, may affect splicing & transcription  

      Bioinformatics: extracting biological information from (huge) data sets [e.g., NCBI GenBank resource]
           Ex.: Analysis of chimpanzee Caspase-12 gene:                 
                        Search for  " Pan + Caspase 12 "
                        Go to  Nucleotide database: Retrieve chimp CASP12 DNA sequence in FASTA formsat
                         Identify human homolog human CASP12 (variant 2) with BLAST [nucleotide collection]                        
                         Find ORFs in chimp & human sequences with ExPASy: what's up with that?

            Ex.: Species identification via mtDNA forensics: "How To Tell a Sea Monster"

                     NADH2 sequence identified from GenBank


    Proteomics: the total pattern of protein expression in an organism
            There is one genome, but many proteomes:
             DNA Chips to explore life-stage & tissue-specific patterns

All text material ©2012 by Steven M. Carr