ch 12. researchers can insert desired genes into plasmids, creating recombinant dna and insert those...
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Researchers can insert desired genes into plasmids, creating recombinant DNA and insert those plasmids into bacteria
Bacterium
Bacterialchromosome
Plasmid
1 Plasmidisolated
3 Gene insertedinto plasmid
2 DNAisolated
Cell containing geneof interest
DNAGene ofinterest
Recombinant DNA(plasmid)
4 Plasmid put intobacterial cell
Recombinantbacterium
5 Cell multiplies withgene of interest
Copies of proteinCopies of gene
Clone of cellsGene for pestresistanceinserted intoplants
Gene used to alter bacteriafor cleaning up toxic waste
Protein used to dissolve bloodclots in heart attack therapy
Protein used tomake snow format highertemperature
Figure 12.1
Creating recombinant DNA using restriction enzymes and DNA ligase
Restriction enzymerecognition sequence
G A A T T CC T T A A GDNA1
2
3
4
C T T A AA AT TC
A AT TCG
C T T A AAddition of a DNAfragment fromanother source
Two (or more)fragments sticktogether bybase-pairing
G A AT T CC T TA A G
G A AT T CC T TA A G
5
DNA ligasepastes the strand
Restriction enzymecuts the DNA intofragments
Recombinant DNA molecule
GG
Sticky end
G
Figure 12.2
Cloning a gene in a bacterial plasmid
1Isolate DNAfrom two sources
2Cut both DNAswith the samerestriction enzyme
E.coli
PlasmidDNA
Gene V
Sticky ends
3 Mix the DNAs;they join bybase-pairing
4 Add DNA ligaseto bond the DNA covalently
5 Put plasmid into bacteriumby transformation
Gene VRecombinant DNAplasmid
Recombinant bacterium
6 Clone the bacterium
Bacterial clone carrying manycopies of the human gene
Human cell
Figure 12.3
Cell nucleus
Isolation of mRNAand addition of reversetranscriptase; synthesisof DNA strand
RNA splicing2
Transcription1
3
Breakdown of RNA4
Synthesis of secondDNA strand
5
mRNA
DNA ofeukaryoticgene
IntronExon
RNAtranscript
Exon Intron Exon
Reverse transcriptaseTest tube
cDNA strandbeing synthesized
cDNA of gene(no introns)
Reverse transcriptase makes single stranded DNA from mRNA
cDNA
Therapeutic hormones & vaccines– In 1982, humulin, human insulin produced by
bacteria• Became the first recombinant drug approved by the
Food and Drug Administration
– Vaccines are harmless mutants or derivative of a pathogen that stimulate the immune system
Figure 12.7A
• GM organisms– Acquired genes by artificial means– Transgenic organisms
Agrobacterium tumefaciens
DNA containinggene for desired trait
Tiplasmid
Insertion of geneinto plasmid
RecombinantTi plasmid
1
Restriction site
Plant cell
Introductioninto plantcells
2
DNA carrying new gene
Regenerationof plant
3
Plant with new trait
Gene therapy (or the alteration of an afflicted individual’s genes) may someday help treat a variety of diseases
Cloned gene(normal allele) 1 Insert normal gene
into virus
2 Infect bone marrowcell with virus
3 Viral DNA insertsinto chromosome
4 Inject cellsinto patient
Bonemarrow
Bone marrowcell from patient
Viral nucleicacid
Retrovirus
Figure 12.13
• DNA profiling
Crime sceneDNA isolated1
Suspect 1 Suspect 2
DNA of selectedmarkers amplified
2
Amplified DNA compared
3
Cycle 1yields 2 molecules
21 3
GenomicDNA
Cycle 3yields 8 molecules
Cycle 2yields 4 molecules
3 5 3 5 3 5
Targetsequence
Heat toseparateDNA strands
Cool to allowprimers to formhydrogen bondswith ends oftarget sequences
35
3 5
35
35 35
Primer New DNA
5
DNApolymerase addsnucleotidesto the 3 endof each primer
5
PCR used to amplify target section of DNA
Gel electrophoresis sorts DNA molecules by size
+ +
– –
Powersource
Gel
Mixture of DNAmolecules ofdifferent sizes
Longermolecules
Shortermolecules
Completed gel
Figure 12.10
– After digestion by restriction enzymes the fragments are run through a gel
–
+
Longerfragments
Shorterfragments
x
w
y
z
y
1 2
Figure 12.11B
DNA fingerprinting can help solve crimes
Defendant’sblood
Blood fromdefendant’s clothes Victim’s
blood
Figure 12.12A Figure 12.12B
• STR– Short sequences of DNA repeated many
times in a row– STR analysis compared lengths of STR
sequences at specific sites on the genome
• Used in forensic investigations
STR site 1
Crime scene DNA
STR site 2
Suspect’s DNA
Number of short tandemrepeats match
Number of short tandemrepeats do not match