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DNA Techniques. Recombinant DNA DNA Sequencing Gel Electrophoresis DNA Amplification DNA Fingerprinting Gene Therapy. by no means all. Recombinant DNA. Getting genes from one organism into another Target gene vs. vector Restriction endonucleases tool for cutting DNA prior to insertion.

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dna techniques
DNA Techniques
  • Recombinant DNA
  • DNA Sequencing
  • Gel Electrophoresis
  • DNA Amplification
  • DNA Fingerprinting
  • Gene Therapy

by no means all...

recombinant dna
Recombinant DNA
  • Getting genes from one organism into another
  • Target gene vs. vector
  • Restriction endonucleases
    • tool for cutting DNA prior to insertion
restriction endonucleases
Restriction Endonucleases
  • Naturally occurring bacterial enzymes
    • Cut DNA (of attacking viruses?)
    • break phosphodiester bonds
  • Named for organism of origin
    • EcoRI came from E. coli strain RI
restriction endonucleases4
Restriction Endonucleases
  • “Restriction” = restricted to cutting at very specific sites
    • palindromic DNA sequences
    • inverted repeats
    • flush cutters and staggered cutters
sample of restriction enzymes and cutting sites
Sample of Restriction Enzymes and Cutting Sites

EcoRI 5’G A A T T C

C T T A A G 5’

Staggered cut

EcoRII 5’ G C C T G G C

C G G A C C G 5’

Staggered cut

staggered cuts vs flush cuts

A A G C T T

T T C G A A

5’ A A G C T T

T T C G A A 5’

Staggered Cuts vs. Flush Cuts

5’ G T Py Pu A C

C A Pu Py T G 5’

G T Py Pu A C

C A Pu Py T G

"sticky ends"

sticky ends
Sticky Ends
  • Unpaired bases have affinity for other unpaired bases
    • will reform hydrogen bonds w/ complementary bases
slide8

--C G C T A C C A A G C T T A C C G A--

--G C G A T G G T T C G A A T G G C T--

--G C T A C C A A G C T ----- A G C T T A C C G --

--C G A T G G T T C G A ----- T C G A A T G G C --

A G C T --------

-------- T C G A

Recombinant DNA

cloning
Cloning
  • Making multiple copies of a target gene
  • Generally use bacteria as the “factory”
    • reproduce incredibly quickly
    • have natural VECTOR
      • Vectors carry the target gene
cloning10
Cloning
  • Vectors
    • segments of DNA that carry target gene; allow reproduction of gene
      • plasmids
      • phages
      • cosmids
        • part viral, part plasmid
      • BACs - bacterial artificial chromosomes
      • YACs - yeast artificial chromosomes
cloning11
Cloning

chromosome

Target gene

Need restriction enzyme site on either side of target gene

as well as w/in vector!

Vector

cloning12
Cloning
  • Use SAME restriction endonuclease to cut out target gene and open vector
  • Must use an enzyme that produces sticky ends!
  • Will have complementary sticky ends on target and vector
cloning13
Cloning

Bacteria will now produce target gene product

Transform bacteria w/ vector

cloning14
Cloning
  • Interferon was the first human protein to be cloned this way
  • Human proteins produced w/ recombinant DNA technology:
    • insulin
    • human growth hormone
    • superoxide dismutase (heart attack)
    • Factor VIII (hemophilia)
slide15
Human proteins produced w/ recombinant DNA technology:

Fertility hormones

Interleukin-2 (kidney disease)

erythropoietin (anemia)

lung surfactant protein (premature infants)

DNase (cystic fibrosis)

Renin inhibitor (high blood pressure)

colony stimulating factor (post chemo-therapy)

transgenic organisms
Transgenic organisms
  • Genes may be transferred from eukaryotic one organism to another
    • plants that glow in the dark
      • transferred luciferase gene
    • plants w/ herbicide resistance
    • cows w/ human lactoferrin gene
      • binds iron, prevents infection, (infant formula)
    • goats w/ human tissue plasminogen activator
      • breaks up blood clots
gel electrophoresis
Gel Electrophoresis
  • Used to separate molecules
    • Protein, RNA, DNA
    • Separation based on size of molecules, charge, shape, etc.
  • Necessary for Southern blots, DNA sequencing, DNA fingerprinting, RFLP analysis, many other applications
slide18

-

+

protein gels
Protein gels
  • Used to identify genotypes
  • Electrophoretic patterns inherited as alleles
    • Electromorphs or allelomorphs
    • A : one protein
    • a : slightly different protein
protein gels20
Protein gels

1 2 3

Lane 1: homozygote for slow allele

Lane 2: heterozygote for both alleles

Lane 3: homozygote for fast allele

rflps restriction fragment length polymorphisms
RFLPs:Restriction Fragment Length Polymorphisms
  • Differences in restriction sites; DNA from different sources cut into different sizes
    • Flush or staggered cutters
  • Quantify differences between species; relatedness
  • Identify source of tissue samples (forensics, etc.)
  • Markers for genetic disease
rflps as markers
RFLPs as markers

Normal b-globin gene:

Mst II site

Sickle cell b-globin gene:

no restriction site

rflps as markers27
RFLPs as markers

Ind. A Ind. B

SS Ss ss

southern blots
Southern Blots
  • Way to visualize specific DNA segments
  • Transfers DNA from gel to nitrocellulose filter
  • “Stains” specific DNA segment w/ radioactive, single-stranded DNA probe
    • Northern blots: RNA
    • Western blots: protein
southern blot
Southern Blot

If stain for non-specific

DNA, see a “smear”

1. Heat to separate strands

2. Add radioactive probe

3. Cool, probe binds to

complementary areas

4. Place filter on X-ray film,

develop, see bands!

dna fingerprinting
DNA Fingerprinting
  • Very specific form of RFLP analysis
    • allows differentiation even if no differences in restriction sites
  • Uses VNTRs
    • variable number of tandem repeats
    • short, repeated sequences (~ 4 bases)
      • GGACGGACGGACGGACCC TGCCTGCC TGCCTG
    • non-coding, highly variable between individuals
dna fingerprinting parentage tests
DNA Fingerprinting - Parentage tests

mother

child

Mr. A Mr. B Mr. C

dna sequencing
DNA Sequencing
  • Sanger or Dideoxynucleotide method
  • Uses base analogues that are “chain terminators”
  • Depends on ability of electrophoresis to distinguish between DNA segments that differ in length by only one base
dna sequencing33
DNA Sequencing
  • Dideoxynucleotides
    • sugar missing two oxygens
      • oxygen on 3’ carbon missing
        • no new nucleotide may bind
          • chain terminator
  • Copy DNA template using ddnucleotides
dna sequencing34
DNA Sequencing
  • Run four DNA synthesis reactions
    • one with 4 normal bases and some ddATP
    • one with 4 normal bases and some ddTTP
    • one with 4 normal bases and some ddCTP
    • one with 4 normal bases and some ddGTP
  • Run a gel; each rxn in a lane (4 lanes)
slide35

3’---------T A C T A T G C C A G A 5’

5’ primer

ddATP rxn:

5’ primer A T G A T A

5’ primer A T G A

(Sometimes the entire segment is replicated. This

happens in all the rxns and can be ignored.)

5’ primer A

Three different segments produced: 1 base, 4 bases,

and 6 bases in length ignoring primer.

slide36

3’---------T A C T A T G C C A G A 5’

5’ primer

ddTTP rxn:

5’ primer A T G A T A C G G T C T

5’ primer A T G A T A C G G T

5’ primer A T G A T

5’ primer A T

slide37

3’---------T A C T A T G C C A G A 5’

ddATP

A T G A T A

A T G A

A

ddTTP

A T G A T A C G G T C T

A T G A T A C G G T

A T G A T

A T

ddGTP

A T G A T A C G G

A T G A T A C G

A T G

ddCTP

A T G A T A C G G T C

A T G A T A C

slide38

3’---------T A C T A T G C C A G A 5’

“Read” sequence:

Run on a gel:

T

C

T

G

G

C

A

T

A

G

T

A

5’

A T G A T A C G G T C T

A T G A T A C G G T C

A T G A T A C G G T

A T G A T A C G G

A T G A T A C G

A T G A T A C

A T G A T A

A T G A T

A T G A

A T G

A T

A

polymerase chain reaction pcr
Polymerase Chain Reaction (PCR)
  • Used to amplify DNA samples
  • Small tissue sample --> enough DNA to do fingerprinting, etc.
  • Repeated cycles of DNA replication
  • Uses Taq polymerase
slide40
PCR

heat

Cool in presence of

primer (DNA oligonucleotide)

nucleotides, polymerase

Repeat cycle many times