Chapter 19 molecular genetic analysis and biotechnology
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Chapter 19 – Molecular Genetic Analysis and Biotechnology. Recombinant DNA technology. One molecule composed of two distinct DNA sources Biotechnology Development of commercial products; medical applications. Restriction endonucleases/ enzymes. Make double-stranded cuts in DNA

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Chapter 19 – Molecular Genetic Analysis and Biotechnology

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Chapter 19 molecular genetic analysis and biotechnology

Chapter 19 – Molecular Genetic Analysis and Biotechnology


Recombinant dna technology

Recombinant DNA technology

  • One molecule composed of two distinct DNA sources

  • Biotechnology

    • Development of commercial products; medical applications


Restriction endonucleases enzymes

Restriction endonucleases/ enzymes

  • Make double-stranded cuts in DNA

  • Bacterial source – guards against viral invasion

    • Bacterial DNA is methylated; viral unmethylated

  • Name of enzymes is an abbreviation of bacterial source

  • Usually recognizes 4-6 pallindromic sequences


Digestion

Digestion

  • Blunt ends

    • Cut both strands of DNA at same location

  • Sticky/cohesive ends

    • Produce staggered cuts; single stranded “sticky” ends

    • Any DNA cut with the same enzyme will have ends with the same sequence

      • Can combine DNA from different sources and seal cuts with enzyme ligase


Gel electrophoresis

Gel electrophoresis

  • Porous gel made of agarose or polyacrylamide

  • Sample DNA mixed with loading dye that allows for visualization and increases density

  • Negatively-charged DNA runs toward positive pole when electrical current passes through the gel

  • Separates fragments based on size

    • Smaller fragments migrate the furthest – bottom of the gel

  • Ladder or marker contains fragments of known sizes to aid in determination of sample fragment size

  • Expose gel to dye

    • Methylene bue – light box

    • Ethidium bromide – UV light


Southern blotting dna

Southern blotting - DNA

  • Restriction digestion of genomic DNA and separated by gel electrophoresis

    • Large number of band sizes produce smear on gel

  • Fragments are denatured into single-strands and transferred from gel to a thin nylon or nitrocellulose membrane


Southern blotting con t

Southern blotting con’t

  • Membrane is exposed to probe that has been radioactively- or fluorescently labeled

    • Probe has complementary sequence to target sequence

  • Unbound probe is rinsed away and bound probe is detected

  • Northern blotting – RNA

  • Western blotting - protein


Cloning genes

Cloning genes

  • Produces duplicate copies of specific genes

    • Provides large number of copies

  • Insert gene of interest into bacterial cells for rapid replication


Cloning vector

Cloning vector

  • DNA gene of interest is inserted into a cloning vector

  • Requirements:

    • Origin of replication

    • Unique restriction site

      • Has only one recognition site

    • Selectable marker

      • Antibiotic resistance


Chapter 19 molecular genetic analysis and biotechnology

LacZ

  • Intact plasmid

    • Ampillicin resistance

    • Β-galactosidase cleaves X-gal and bacteria is blue

  • Recombinant plasmid

    • Ampillicin resistance

    • Inserted sequence disrupts β-galactosidase gene; bacteria remains white


Expression vectors

Expression vectors

  • Used not just for copies of gene, but to make gene product

    • Gene expression

  • Requires sequences for transcription/ translation


Cloning vectors

Cloning vectors

  • YACs

    • Yeast artificial chromosomes

    • Yeast origin of replication, centromere, telomeres

    • ~600kb – 1,000kb

  • BACs

    • Bacterial artificial chromosomes

    • ~100-500kb

  • Shuttle vectors

    • Can be transferred between two different species (bacteria and yeast)

    • Origin of replication and markers must be recognized by both organisms


Polymerase chain reaction pcr

Polymerase Chain Reaction (PCR)

  • Amplifies DNA fragments in vitro

  • Taq polymerase

    • Isolated from hot spring bacteria Thermus aquaticus

    • stable at near boiling temperatures

  • Automated thermocyclers

    • Computer aided machine that rapidly changes temperature


Pcr needed components

PCR needed components

  • Target DNA

  • Primers – 2 different (one for each strand)

    • Complementary to end sequences

  • dNTPs

  • Buffer/Mg ions

  • Polymerase


Pcr steps

PCR steps

  • Denaturation

    • Separates DNA into single strands

    • ~90°C

  • Annealing

    • Primers complementary pair to DNA strands

    • ~55°C

  • Elongation/extension

    • Polymerase adds new nucleotides to primers’ 3′ end

    • ~72°C


Pcr con t

PCR con’t

  • Produces billions of copies of target DNA in a few hours

  • Reverse transcription PCR

    • Makes cDNA from RNA template

  • Real-time PCR

    • Quantifies amount after each cycle

    • Allows measurement of mRNA; amount of gene expression

  • Limitations

    • Need to know DNA sequence – at least the ends

    • Contamination gets amplified as well

    • Taq polymerase has no proofreading capabilities

      • Newer polymerases do

    • Limited to small sizes (less than 2,000kb)


Gel electrophoresis results

Gel Electrophoresis Results


Restriction fragment length polymorphisms rflps

Restriction Fragment Length Polymorphisms (RFLPs)

  • Variation from individual to individual

  • Helps with linkage studies for gene mapping

  • DNA fingerprinting

    • Also uses microsatellites – short tandem repeats

      • Size of fragment depends on number of repeats


Dna sequencing

DNA sequencing

  • Dideoxy sequencing

  • Normal nucleotides dNTPs – deoxyribonucleoside triphosphate

  • ddNTPs – dideoxyribonucleoside triphosphate

    • Missing the oxygen at the 3′ carbon

    • No nucleotide can be added to strand


Dna sequencing con t

DNA sequencing con’t

  • 4 reaction tubes are set up – one for each base

  • DNA is then denatured and run on a gel


Dna sequencing con t1

DNA sequencing con’t

  • Sequence on gel is complementary to original strand

  • Automated sequencers use ddNTPs labeled with fluorescent dye

    • Sample is analyzed by a computer and sequence is graphed out


Applications

Applications

  • Pharmaceuticals

    • Bacterial production of human insulin, growth hormone

  • Bioremediation

    • Bacteria genetically engineered to break down toxic chemicals

  • Agriculture

    • Viral/pesticide resistance; increase nutritional value


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