DNA is purified from cells or tissues

1
Basic Steps

• DNA is purified from cells or tissues
• DNA is cut with restriction enzymes
• DNA fragment are inserted into vectors
• The recombinant DNA molecule is inserted into a
  host cell for
• replication
• Daughter host cells contain recombinant
  molecule
• The cloned DNA is recovered from host cells for
  analysis
• Cloned DNA can be transcribed and translated

2
Restriction Enzymes

• First discovered in E.coli as protection from
  invading DNA
• Enzymes recognize specific sites to be cut on
  the DNA molecule
• DNA sequences are palindromic meaning that the
  sequence is the same if read 5-3 or 3-5
• Some enzymes leave blunt ends, others leave
  sticky ends
• Sticky ends have the ability to associate with
  a complement sequence

Eco RI
Hind III
Pst I
3
Basic Steps

• DNA is purified from cells or tissues
• DNA is cut with restriction enzymes
• DNA fragment are inserted into vectors
• The recombinant DNA molecule is inserted into a
  host cell for
• replication
• Daughter host cells contain recombinant
  molecule
• The cloned DNA is recovered from host cells for
  analysis
• Cloned DNA can be transcribed and translated

4
Vectors Are Carriers of DNA Fragments

• Vectors must replicate independently and carry a
  selectable marker to distinguish from cells not
  containing an insert
• Contain many restriction sites present only
  once in the vector
• DNA fragments with sticky ends can re-anneal
  with sticky ends in the cut vector
• DNA ligase seals the fragments into the vector
  to complete the recombinant DNA molecule
• It should be easily recoverable from the host
  cell

5
Plasmid Vectors

• Naturally occurring, double stranded circular
  DNA extrachromosomal elements
• Plasmids are small which allow insertion of
  large pieces of DNA
• Can reach copy numbers from 10 to 1000 per cell
  
• Large number of unique restriction sites in a
  cluster called a multiple cloning site (MCS)
• Many have antibiotic resistance as selectable
  markers such as ampicillin, kanamycin,
  tetracycline

6
Basic Steps

• DNA is purified from cells or tissues
• DNA is cut with restriction enzymes
• DNA fragment are inserted into vectors
• The recombinant DNA molecule is inserted into a
  host cell for
• replication
• Daughter host cells contain recombinant
  molecule
• The cloned DNA is recovered from host cells for
  analysis
• Cloned DNA can be transcribed and translated

7
Cloning into Plasmids
8
Basic Steps

• DNA is purified from cells or tissues
• DNA is cut with restriction enzymes
• DNA fragment are inserted into vectors
• The recombinant DNA molecule is inserted into a
  host cell for
• replication
• Daughter host cells contain recombinant
  molecule
• The cloned DNA is recovered from host cells for
  analysis
• Cloned DNA can be transcribed and translated

9
Colony Screening Grunstein-Hogness Method
10
Restriction Mapping

• Using the relative sizes of fragments
  produced by digestion with a collection of
  enzymes we can assemble a map of the individual
  restriction sites
• Establish the uncut size
• -Cut with enzyme 1 - run on gel
• -Cut with enzyme 2 - run on gel
• Propose mapping distances based on
  individual cut sites
• Cut with a mixture of 1 and 2 to determine
  alignment

11
An Example of Restriction Mapping a Plasmid
12
An Example of Restriction Mapping a Plasmid
13
Using Restriction Maps to Characterize Clones
Contigs!
14
Preparing DNA for Southern Analysis
Restriction Enzyme
Restriction Enzyme


Sample 2
Sample 1
15
Gel Electrophoresis
16
Schematic Representation of Gel
17
Southern Blotting
18
Labeling a Probe
Cloned DNA
19
Southern Analysis
Detection of Yp sequences in 46 XX males.
20
Sanger Sequencing

• use DNA polymerase to copy a template from a
  primer
• originally used single stranded vector, now use
  double stranded DNA
• add a small amount of nucleotides that cant
  form the 3 bond -dideoxynucleotide
  terminators
• Do 4 separate reactions for each dNTP including
  radioactive or fluorescent labeled dNTP

ddNTP
dNTP
21
Sanger Sequencing
22
Sanger Sequencing
ddATP
ddCTP
ddGTP
ddTTP
23
Automated Sequencing
The template DNA is supplied with a mixture of
all four normal (deoxy) nucleotides in ample
quantities dATP dGTP
dCTP dTTP a mixture of all four
dideoxynucleotides, each present in limiting
quantities and each labeled with a "tag" that
fluoresces a different color ddATP
ddGTP ddCTP ddTTP
24
Chain Termination Also Helps Fight HIV
AZT, a drug used to treat AIDS, is taken up by
cells where it is converted into a triphosphate.
The reverse transcriptase of the human
immunodeficiency virus (HIV) prefers AZT
triphosphate to the normal nucleotide (dTTP). AZT
has no 3' -OH group, so DNA synthesis by reverse
transcriptase halts when AZT triphosphate is
incorporated in the growing DNA strand.
Fortunately, the DNA polymerases of the host cell
prefer dTTP, so side effects from the drug are
not so severe as might have been predicted.
25
Northern Analysis

• Instead of DNA which is used in Southerns,
  Northern Analysis uses mRNA.
• Is used to analyze the where, when, and how
  much of gene expression.

26
Northern Analysis of Adrenal 3bHSD mRNA in
Response to Testosterone Treatment