Ch 12

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Ch 12

• DNA Technology and Genomics

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DNA Technology

• Methods for studying and manipulating genetic
  material
• Cloning
• Genetically Modified
• Gene Therapy
• DNA Profiling
• Genomics

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Recombinant DNA

• Combining nucleotide sequences from 2 sources to
  form a single DNA
• Bacteria often used
• Plasmid

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Creating Recombinant DNA

• Restriction Enzymes
• Restirction site
• Sticky Ends
• Ligase

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Cloning the Gene

• Bacteria containing the gene are cloned
• Typically, antibiotic resistance genes are also
  inserted
• Bacteria are grown on antibiotic medium
• All bacteria without the resistance (and target)
  gene die
• Only those with gene reproduce
• Stored in genomic library
• Plasmids used to store genetic information

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Uses

• Gene Cloning
• Produces multiple copies of gene-carrying DNA
• Genetic Engineering
• Direct manipulation of genes

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cDNA

• Express Eukaryotic genes in Prokaryotic cells
• What about introns?
• Prokaryotes dont have machinery to splice
• mRNA
• Already spliced
• Work from mRNA to create cDNA

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Fig. 12-4
Cell nucleus
Intron
Exon
Exon
Intron
Exon
DNA of eukaryotic gene
Transcription
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RNA transcript
RNA splicing
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mRNA
Isolation of mRNA and addition of
reverse transcriptase synthesis of DNA strand
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Test tube
Reverse transcriptase
cDNA strand being synthesized
Breakdown of RNA
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Synthesis of second DNA strand
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cDNA of gene (no introns)
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Uses

• Diagnosis and treatment of disease
• Vaccines
• Therapeutic hormones

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Probes

• Genomic Library
• How to find the right gene?
• Nucleic Acid Probe
• Locate specific gene or nucleotide sequence

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Probes

• Synthesize short sequence of singe stranded DNA
  of complimentary sequence
• Label radioactive tag
• Mix with genomic library
• Bacteria with gene of interest will glow

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Fig. 12-5
Radioactive DNA probe
Mix with single- stranded DNA from genomic library
Single-stranded DNA
Base pairing indicates the gene of interest
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Genetically Modified

• Organisms who have acquired genes by artificial
  means
• If from another species transgenic organism

Agrobacterium tumefaciens
Plant cell
DNA containing gene for desired trait
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2
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Ti plasmid
Recombinant Ti plasmid
Introduction into plant cells
Regeneration of plant
Insertion of gene into plasmid
DNA carrying new gene
Plant with new trait
Restriction site
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Gene Therapy

• Altering an afflicted persons genes for
  therapeutic purposes
• Treatment of genetically based disorders
• Technology still in infancy

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Fig. 12-10
Cloned gene (normal allele)
Insert normal gene into virus
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Viral nucleic acid
Retrovirus
Infect bone marrow cell with virus
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Viral DNA inserts into chromosome
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Bone marrow cell from patient
Bone marrow
Inject cells into patient
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DNA Profiling

• Forensics
• Scientific analysis of evidence
• DNA Profiling
• Analysis of DNA fragments
• Determine if they originate form a particular
  individual
• PCR
• Gel Electrophoresis

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PCR

• 3 step cycle
• Heat separates strands
• Cooling allows primers to form H bonds with end
  of target sequence
• DNA Polymerase adds nucleotides
• Creates double stranded DNA
• Repeat

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PCR

• Significant breakthrough in genetics
• Original polymerase was from E. coli
• Heating traditionally denatured polymerase
• New polymerase added after each heating cycle
• Giant pain in the butt

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PCR

• Discovery of T. aquaticus
• Lives in hotsprings
• Not denatured under high temps
• Could now be done at higher temps
• Better success rates

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PCR
Cycle 1 yields 2 molecules
Cycle 3 yields 8 molecules
Cycle 2 yields 4 molecules
Genomic DNA
3?
5?
3?
5?
3?
5?
5?
DNA polymerase adds nucleotides to the 3? end of
each primer
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3
Heat to separate DNA strands
Cool to allow primers to form hydrogen bonds with
ends of target sequences
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3?
5?
3?
5?
Target sequence
5?
3?
5?
3?
5?
3?
5?
Primer
New DNA
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Gel Electrophoresis

• Use of gel to separate DNA strands by size
  (molecular weight) or charge
• DNA must first be digested
• Strands must be cut into different sizes
• Use Restriction Enzymes
• Cut DNA in specific places
• Looks for specific nucletoide sequences

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Gel Electrophoresis
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Gel Electrophoresis

• STR
• Short sequences of DNA repeated many times in a
  row
• STR analysis compared lengths of STR sequences at
  specific sites on the genome
• Create a genetic profile of individuals
• STRs differ among individuals

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Gel Electrophoresis

• RFLP
• Restriction fragment length polymorphisms
• Difference between two samples of homologous DNA
  arising from differing locations of restriction
  sites

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• After digestion by restriction enzymes the
  fragments are run through a gel

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• DNA profiling

Crime scene
Suspect 1
Suspect 2
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DNA isolated
DNA of selected markers amplified
2
Amplified DNA compared
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• Used in forensic investigations

STR site 1
STR site 2
Crime scene DNA
Number of short tandem repeats match
Number of short tandem repeats do not match
Suspects DNA
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Genomics

• Studying the entire genome and their interactions

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Human Genome Project

• identify all the approximately 20,000-25,000
  genes in human DNA,
• determine the sequences of the 3 billion chemical
  base pairs that make up human DNA,
• store this information in databases,
• improve tools for data analysis,
• transfer related technologies to the private
  sector, and
• address the ethical, legal, and social issues
  (ELSI) that may arise from the project.

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Fig. 12-18
Exons (regions of genes coding for protein or
giving rise to rRNA or tRNA) (1.5)
Introns and regulatory sequences (24)
Repetitive DNA that includes transposable elements
and related sequences (44)
Unique noncoding DNA (15)
Repetitive DNA unrelated to transposable elements
(15)
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HGP

• 3.2 billion nucleotide pairs
• 21000 genes
• How are we so complex?
• RNA splicing
• Non-coding
• Repetitive DNA
• Telomeres
• Transposable elements

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Shotgun Method

1. Chop up genome
2. Squeeze it through a small, pressurized syringe
3. Clone fragments
4. Insert them into a vector
5. Sequence
6. Search for overlapping segments
7. Reassemble the overlaps

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Chromosome
Chop up with restriction enzyme
DNA fragments
Sequence fragments
Align fragments
Reassemble full sequence
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• Recombo DNA
• http//www.youtube.com/watch?v-sI5vy-cD2gfeature
  related
• PCR
• http//www.youtube.com/watch?v2KoLnIwoZKUfeature
  related
• PCR and Gel
• http//www.youtube.com/watch?v_uoPkavtXgs

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Videos

• Ch Overview
• http//www.youtube.com/watch?vNc3jArZXHjs