cutting and pasting dna the cutters are called restriction enzymes, they cut dna at specific...
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Cutting and Pasting DNA
• The cutters are called restriction enzymes, they cut DNA at specific nucleotide sequences
Restriction enzymecuts DNA intofragments
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Addition of a DNAfragment fromanother source
Fragments sticktogether bybase pairing
DNA ligasejoins the strands
Recognition sequence for a restriction enzyme
Sticky end
Sticky end
Recombinant DNA molecule
How Do I Obtain the Gene of Interest?
• The “shotgun” approach - millions of recombinant plasmids are produced; a genomic library
• How a DNA probe tags a gene
Radioactive probe (DNA)
Single-stranded DNA
Mix with single-stranded DNA from various bacterial clones
Base pairing indicatesthe gene of interest
• The reverse transcriptase method
Transcription1
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Introns removed and exons spliced together
Isolation of mRNA from cell and addition of reverse transcriptase
5 Synthesis of secondDNA strand
Synthesis of cDNAstrand
Exon Intron Exon Intron Exon
Cell nucleus
DNA ofeukaryoticgene
RNAtranscript
mRNA
Cytoplasm
Reversetranscriptase
Test tube
cDNA strandbeing synthesized
cDNA ofgene with-out introns
• DNA technology revolutionized forensics
WHO ARE YOU?
– forensics is the scientific analysis ofevidence from crime scene investigations
• DNA fingerprinting has been available since 1986
• Used in CSI, victim IDs; Cheddar Man
Crimes and Other Investigations
• Polymerase Chain Reaction (PCR) copies DNA
DNA Fingerprinting Techniques - PCR
• DNA amplification by PCR
Tinyamountof DNA
8421
Number of DNA molecules
• DNA fragments cut by restriction enzymes are seen as “bands” in an electrophoresis gel
Longerfragments
Shorterfragments
CrimesceneDNA
SuspectDNA
Defendant’sblood
Blood on defendant’sclothes
Victim’sblood
• The science of studying entire genomes
GENOMICS
• In 1990, an international consortiumbegan the Human Genome Project
The Human Genome Project
– sequencing the human genome
– challenges: large size, and the fact that only a small amount of our DNA is genetic
• Genome mapping
Chop up withrestriction enzyme
Chromosome
Sequencefragments
DNA fragments
Alignfragments
Reassemblefull sequence
• Gene therapy cures disease by altering human genes
HUMAN GENE THERAPY
Normal humangene isolatedand cloned
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Human geneinsertedinto virus
Virus injectedinto patient
Healthy individual
Nonharmfulvirus (vector)
Virus containingnormal human gene
Bone marrow
• Great power brings both promise and danger
SAFETY AND ETHICAL ISSUES
– strict laboratory safety procedures are used
– negotiators from 130 countries agreed on a Biosafety Protocol for GM foods
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