Ch 12

1
Ch 12
2

• Researchers can insert desired genes into
  plasmids, creating recombinant DNA and insert
  those plasmids into bacteria

0
Bacterium
Cell containing gene of interest
Plasmid
Bacterial chromosome
DNA
Recombinant DNA (plasmid)
Gene of interest
Recombinant bacterium
Copies of gene
Copies of protein
Clone of cells
Gene for pest resistance inserted into plants
Protein used tomake snow format
highertemperature
Figure 12.1
Gene used to alter bacteria for cleaning up toxic
waste
Protein used to dissolve bloodclots in heart
attack therapy
3

• Creating recombinant DNA using restriction
  enzymes and DNA ligase

Restriction enzymerecognition sequence
G A A T T C C T T A A G
DNA
1
Restriction enzymecuts the DNA intofragments
A AT TC
G
C T T A A
G
2
Sticky end
A AT TC G
Addition of a DNAfragment fromanother source
G
3
C T T A A
Two (or more)fragments sticktogether
bybase-pairing
G A AT T C C T TA A G
G A AT T C C T TA A G
4
DNA ligase pastes the strand
5
Figure 12.2
Recombinant DNA molecule
4

• Cloning a gene in a bacterial plasmid

0
E.coli
Human cell
Plasmid
DNA
Gene V
Sticky ends
Gene V
Recombinant DNAplasmid
Recombinant bacterium
Figure 12.3
Bacterial clone carrying manycopies of the human
gene
5
Cell nucleus
Intron
Exon
Exon
Intron
Exon
DNA of eukaryotic gene
Reverse transcriptase makes single stranded DNA
from mRNA cDNA
Transcription
1
RNA transcript
RNA splicing
2
mRNA
Isolation of mRNA and addition of
reverse transcriptase synthesis of DNA strand
3
Reverse transcriptase
Test tube
cDNA strand being synthesized
Breakdown of RNA
4
cDNA of gene (no introns)
Synthesis of second DNA strand
5
6

• Recombinant cells and organisms can mass-produce
  gene products

0
7

• 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

0
8

• GM organisms
• Acquired genes by artificial means
• Transgenic organisms

Agrobacterium tumefaciens
Plant cell
DNA containing gene for desired trait
1
2
3
Ti plasmid
Recombinant Ti plasmid
Introduction into plant cells
Insertion of gene into plasmid
Regeneration of plant
DNA carrying new gene
Plant with new trait
Restriction site
9

• Gene therapy (or the alteration of an afflicted
  individuals genes) may someday help treat a
  variety of diseases

0
Cloned gene (normal allele)
Viral nucleic acid
Retrovirus
Bone marrow cell from patient
Bone marrow
Figure 12.13
10

• DNA profiling

Crime scene
Suspect 1
Suspect 2
1
DNA isolated
DNA of selected markers amplified
2
Amplified DNA compared
3
11
PCR used to amplify target section of DNA
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
2
3
Heat to separate DNA strands
Cool to allow primers to form hydrogen bonds with
ends of target sequences
1
3?
5?
3?
5?
Target sequence
5?
3?
5?
3?
5?
3?
5?
Primer
New DNA
12

• Gel electrophoresis sorts DNA molecules by size

0
13

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

0
14

• DNA fingerprinting can help solve crimes

0
Figure 12.12A
15

• STR
• Short sequences of DNA repeated many times in a
  row
• STR analysis compared lengths of STR sequences at
  specific sites on the genome

16

• 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
17

• How Restriction Fragments Reflect DNA Sequence
• Restriction fragment length polymorphisms (RFLPs)
  reflect differences in the sequences of DNA
  samples

0
Figure 12.11A