Biotechnology and Genomics

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Biotechnology and Genomics
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The Human Genome

• Mitotic (doubled) chromosomes taken from a white
  blood cell at metaphase
• 23 chromosome pairs 46 total
• Chromosomes are arranged by
• size
• location of centromere

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Visualizing DNA

• Polymerase Chain Reaction (PCR)
• Gel Electrophoresis
• DNA fingerprinting
• Genetic testing
• DNA Sequencing
• Characterize genes
• Genetic testing
• Comparing genes among species
• Microarrays (Gene chips)
• Looking at patterns of gene expression

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Polymerase Chain Reaction

• PCR is a technique that allows researchers to
  amplify (make many copies) of a gene.
• Requires only a tiny sample of cells which are
  the source of chromosomes
• PCR is used to generate DNA fingerprints
• Criminology
• Genetic testing
• Parentage

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PCR is based upon DNA Replication

• Helicase unwinds the DNA
• RNA primers are laid down
• DNA polymerase adds nucleotides at the 3 end
• The gaps between new DNA strands is closed in by
  DNA ligase

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The PCR Reaction

• Source of cells (chromosomes)
• DNA nucleotides
• Primers
• DNA polymerase
• Taq polymerase isolated from a bacterium that
  lives in hot springs
• Taq polymerase will not denature at high
  temperatures!

A
nucleotides
C
Taq polymerase
T
primers
G
Chromosomes
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PCR technique

• Process involves alternating cycles of heating
  cooling this mixture.
• Melt denaturing the hydrogen bonds in the DNA
  double helix with heat
• Anneal anneal primers to the single strands of
  DNA
• Copy Taq polymerase copies the DNA
• Repeat

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DNA Gel Electrophoresis

• A technique for separating DNA fragments
• DNA samples amplified by PCR are loaded onto an
  agarose gel
• Agarose is a carbohydrate that forms a gel---- a
  little stiffer than JELLO!
• DNA fragments can travel through the microscopic
  pores in the agarose

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• An electric current is applied to the gel
• DNA fragments separate as they are pulled toward
  the positive electrode
• DNA is negatively charged, so it is attracted to
  the positive electrode
• DNA fragments generated by PCR separate according
  to size
• The shorter fragments move the farthest in the gel

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DNA Fingerprinting

• Requires knowing the sequence of some of the
  genes in the human genome
• Primers are made based on known DNA sequences
• DNA is isolated from skin, hair or blood
• PCR and DNA gel electrophoresis is preformed.

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What genes to use for Fingerprinting?
The Human Genome 30,000 genes, 3 billion bases
We are 99.9 the same
Variable DNA Sequences
account for the 0.01 differences between
individuals of the human species (300,000 bases)
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Short Tandem Repeats (STRs)

• Short tandem Repeats are short sequences of DNA
  that are repeated numerous times in the human
  genome
• The 16 bp sequence GATAGATAGATAGATA is 4 copies
  of the GATA STR.
• The chromosomal locations of STRs are the same
  for every person
• The number of repeats at any given site are
  highly variable for every person.
• PCR can detect these STRs.

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A real DNA fingerprint
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Making cDNA

• Many biotechnologies are based on the mechanism
  for DNA replication and the base pairing rules
• cDNA stands for copy DNA
• cDNA is a copy of a mRNA!
• Scientists use cDNA because the genes for
  proteins contain introns

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Making cDNA from mRNA
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Hybridization

• Relying on the ability to detect the presence of
  genes or mRNA because of the complimentary base
  pair rules
• Hybridization allows single stranded DNA and mRNA
  molecules to attach by base pairing- to
  complimentary base sequences
• The cDNA is often called a DNA probe.

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The Human Genome Project

• Sequencing of the entire human genome
• 46 chromosomes
• 2.9 billion nucleotides
• Government funded consortium and a private
  company called Celera
• Chromosomes from 6-10 anonymous individuals
• Completed in 2003
• Estimate 25,000 genes

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DNA Sequencing

• Knowing the nucleotide sequence of a gene..any
  gene
• Generates databases of DNA sequences that can
  then be used for many applications

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The HGP is not just about humans!

• Knowing the genomes of model organisms
• E. coli
• Yeast
• Fruit fly
• Mouse
• Rhesus monkey
• Chimpanzee

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The human genome project has lead to many new
fields of study

• Bioinformatics
• Storage, retrieval, and comparison of DNA
• Pharmacogenetics
• Customizes drugs to match a persons genetic
  make-up
• Proteomics
• Study of proteins encoded by human genome
• Drug companies can target 500 proteins currently

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Bioinformatics

• Storage of DNA sequences in a computerized
  database
• Retrieval of DNA sequence information
• Comparison of DNA sequences within a given
  species
• Comparison of DNA sequences among different
  species address questions about evolutionary
  relationships

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Comparison of DNA sequences

• A mutation at the 12th nucleotide changed a
  Thymine to an Adenine.
• This mutation distinguishes the Vermont
  population from the other Atlantic coastal
  populations (Maine and New Jersey)

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Comparison of amino acid sequences
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• Evolutionary relationships among primates
• Data is from DNA sequence analyses

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Proteomics

• Study of proteins encoded by human genome
• Combines the words Protein and Genomics
  Proteomics
• Endeavors to know all the proteins coded by the
  human genome
• Deals with the conundrum of how 25,000 genes can
  code for hundreds of thousands of proteins.

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Proteomics Applications for Medicine

• Drug companies can target drugs to 500 proteins
  currently
• Know a proteins shape and amino acid sequence
  allows scientists to make drugs that can fit into
  a protein, for instance, the active site.
• Remember receptor mediated endocytosis!

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Pharmacogenetics

• Customizes drugs to match a persons genetic
  make-up
• In 5-10 years persons may be taking genetic
  screening tests (before doctors write a
  prescription
• DNA microarrays or gene chips

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Gene Chips(Microarray Analysis)

• Discovery of new genes
• Disease Diagnoses
• Drug discovery

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DNA Microarrays or Gene Chips can help customize
a drug regimen.

• Example Lymphoma
• There are different types of Lymphoma depending
  upon which genes are expressed A-F
• Isolate mRNA from the patient
• Make fluorescent cDNA copies of patients mRNA

Actually there are 17 different genes related to
Lymphoma
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• Hybridize the cDNA probes to a gene chip that
  contains all six genes related to Lymphoma
• If the patient has cDNA related to Lymphoma, the
  patient-specific combination of lymphoma genes
  will light-up on the gene chip.
• Doctor can decide on treatment

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• Each dot on a chip represents a unique
  cDNA(gene)
• 17 genes known to be active in B-cell Lymphoma,
  cancer of the white blood cells
• Different subset of the 17 genes is expressed for
  the variations of B-cell lymphoma
• Doctors hybridize mRNA labeled with florescent
  dye from a patient suspected of B-cell lymphoma
  to the cDNA gene chip.
• Where ever the florescent mRNA sticks to the
  cDNA it forms a pattern on the gene chip
• The pattern is read by a computer.
• Doctors can tell which variant of B-cell lymphoma
  the patient has and prescribe the best drug
  regimen

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Dermagenetics

• The Dermagenetics formulation is based on genetic
  testing which detects patterns of gene expression
  regarding dermatology
• The test looks at 5 specific genes that play a
  role in the way the body (skin) responds to types
  of stress
• Photoageing (damage by the sun),
• Skin irritation,
• Environmental stress (damage by chemicals and
  pollutants in your environment).