RECOMBINANT DNA TECHNOLOGY Reference: Ch. 12, pp. 365394

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DNA TECHNOLOGIES
Foundation Techniques Southern Blot
Hybridization, Restriction Enzymes, and DNA
Cloning
Suggested readings2nd edition Ch. 18, pp.
489-5092nd edition Ch. 19, pp. 537-5462nd
edition Ch. 21, pp.577-5853rd edition Ch. 18,
pp. 482-5013rd edition Ch. 19, pp. 521-5383rd
edition Ch. 21, pp.571-580
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Reminder All molecular techniques are based on
the chemical personality (or chemical
properties) of the DNA molecule (or nucleic
acids)
You should therefore revisit the DNA structure
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Negatively-charged phosphate-sugar backbone
Various lengths
Specificity of nucleotides
Hydrogen bonds
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Southern Blot Hybridization
DNA Gel Electrophoresis and the Underlying
Concepts

• Genomics
• Genome sequencing

• Medical Diagnostics

Genetic Engineering

• Restriction Enzymes
• DNA or Gene Cloning and Plasmids
• Gene Therapy

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DNA GEL ELECTROPHORESIS

• Look at the DNA features that this technique
  utilizes.
• 2. Look at the technique itself and what it
  generates.
• 3. How the technique works.

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DNA GEL ELECTROPHORESIS

• For separating DNA strands of any size/length
• Uses a gel to separate DNA strands
• Uses electricity

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DNA GEL ELECTROPHORESIS TAKES ADVANTAGE OF 3 KEY
PROPERTIES OF THE DNA MOLECULE

• The ____________________sugar- phosphate
  backbone.
• The __________________of the DNA molecule, as
  opposed
• to a globular structure.
• 3. The _______________________ behaviour along
  a strand
• of DNA, in other words, chemically alike along a
  DNA strand.

-POLE
-
-
-
-
POLE
PROTEINS
DNA
vs.
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WHAT MAKES GEL ELECTROPHORESIS WORK?

• ____________ both in the DNA molecule and in the
  electrical field.
• THE ________ in the gel and _________ of the
  pores.
• The gel separates DNA strands by
• ________________________________
• Small differences in DNA size can be magnified
  by adjusting the ___________ of the gel, by
  adjusting gel concentration.

Head-on views
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• Examples of Uses
• Diagnostics
• Finding genes

Southern Blot Hybridization
Figure 18.9
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________________________________
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__________ __________ __________
__________ __________ __________
Isolate DNA
To pin the _______to the ___________, evidence
is needed to show that the _____________________
______________________
Digest DNA with restriction enzymes
Make Gel Run Gel
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The gels now become flat gel replicas in the form
of blots
Hybridization experiment
_________
___________
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Hybridize or search with an ________-specific
probe
_________
_________
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Medical Diagnostics - A future visit to the
doctors office
Imagine this, or not?

• Part 1
• Isolate your DNA
• Sequence your entire genome
• Burn the information onto a CD or a flash drive
• Synthesize every gene and dot each onto a
  microchip
• Part 2
• For diagnosis, isolate mRNA samples from biopsies
• Create fluorescence probes
• React with microchip
• Read results and make diagnosis

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• Isolate your DNA
• Sequence your entire genome
• Burn the information onto a CD or a flash drive
• Synthesize every gene and dot each onto a
  microchip

• For diagnosis, isolate mRNA samples from biopsies
• Create fluorescence probes
• React with microchip
• Read results and make diagnosis

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Isolate mRNA samples from biopsies
Cancerous tissue biopsy
Normal tissue biopsy
Create fluorescence probes
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React with microchip
Read results and make diagnosis
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Read the results, compare, and propose a diagnosis
Cancerous cell expression profile
Normal cell expression profile
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Genetic Engineering and Recombinant DNA
Technologies
SNUPPY THE PUPPY is the first cloned canine.
MICE THAT OVEREXPRESS a gene known as Klotho live
longer than their normal brethren, scientists
say. Lab mice typically live only two years, but
the genetically engineered ones pictured here
recently celebrated their third birthdays.
Previous work had found that mice lacking Klotho
die early.
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What is meant by the term RECOMBINANT DNA?

• Bottom line definition The ___________of DNA
  molecules from at least _________________________
  _________

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• Sources can be
• 1. From ______________species
• 2. From __________________-locations
• 3. Different ______ parts
• e.g. promoter and coding sequences

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• Therefore, recombinant DNA production
• is, to a large degree, an artificial or
  deliberate process as opposed to evolutionary
  events.

In order to create recombinant DNA molecules, you
would need to manipulate in a controlled manner
pieces of DNA gtgtgtgtDNA manipulation or DNA cloning
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• DNA cloning is thus a way to manipulate genes
  and a way for copying your designer genes

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Tools for Cloning DNA or Designing Genes
Figure 18-1
The DNA molecule itself
Cutting and gluing enzymes
Copying vehicles or Cloning Vectors
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• 1. The DNA molecule itself
• Properties that allow us to manipulate genes
• DNA is arguably the easiest molecule to work
• with, as opposed to RNA or proteins
• Easy to isolate and separate
• Unique sequences allow us to
• manipulate specific DNA pieces

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• 2. Cutting and gluing enzymes
• (i) ______________ enzymes (endonucleases)
• Enzymes that recognize specific DNA sequences and
  cut the double-stranded DNA molecule at specific
  sites
• (ii) DNA ______(from DNA replication and DNA
  repair)
• Enzyme for re-forming the covalent
  sugar-phosphate backbone

Figure 18-2
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• 3. Copying vehicles or Cloning Vectors
• Vectors are designed to be mini-genomes
• DNA cloning is usually carried out using
  prokaryotic systems
• Commonly-used systems being
• bacterial plasmids
• viral genomes

Figure 18-2
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(No Transcript)
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Properties of Restriction Enzymes Useful for DNA
Cloning

• These enzymes are DNA endonucleases
• Recognize short DOUBLE-STRANDED DNA sequences
• These sequences, in many cases, are called
  PALINDROMES because of the reverse/mirror image
  of the recognition sequences in the DNA strands
• Cut precisely and reproducibly
• The enzymes leave different types of cut-ends
  useful ones being overhangs (2 types) and blunt

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The overhanging ends are single-stranded and
capable of base pairing, generating what we call
STICKY ENDS
DNA END (1)
DNA END (2)
EcoRI
EcoRI
Sticky ends can be used to bring pieces of DNA
together
EcoRI
HindIII
DNA END (4)
DNA END (3)
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CLONING VECTORS

• Vectors are used to carry and to amplify DNA
  fragments
• They also contain selection capabilities to
  help you follow your cloning experiments
• The most useful and common cloning vectors are
  based on BACTERIAL PLASMIDS

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• WHAT ARE PLASMIDS?
• They are miniature bacterial genomes
• They carry a limited number of genes
• Replicate autonomously and independently
• Usually in multiple copies

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• A TYPICAL CLONING VECTOR
• Contains 3 features to facilitate all gene
  manipulation requirements
• Various sites for inserting (Cloning) your DNA
  fragments
• An origin of replication for amplification
• Antibiotic resistant genes for selection
  purposes in order to follow your cloning
  experiment

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Multiple cloning sites
Replication origin
Antibiotic genes for selection work
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_______________ _____________
Figure 18-2
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• Cloning a specific DNA fragment

__________
BamHI
BamHI
SOURCE DNA
BamHI cut
Isolate
BamHI
Mix
Various combinations arise
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BamHI
The DNA fragments are held together by hydrogen
bonds of the STICKY ENDS Add DNA
Ligase This enzyme restores the covalent
sugar-phosphate backbone or bond
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3
3-OH 5-P
5
5
5-P 3-OH
3
Introduce into a plasmid-less bacterial host
All possible combinations will be introduced into
bacteria, so we need to select the right cells.
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• Allow all cells to grow on selective media
• e.g. containing the antibiotic AMPICILLIN

To select plasmids with versus without DNA
inserts
Further testing on 2nd antibiotic TETRACYCLINE
DEAD
ALIVE
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• SUMMARY OF A TYPICAL CLONING PROTOCOL
• 1. Isolate your target DNA
• 2. Cut with the selected restriction enzyme(s)
  and isolate the specific DNA fragment to be
  cloned
• 3. Cut the cloning vector with the same
  restriction enzyme(s)
• 4. Mix (2) and (3), DNA fragment and vector,
  together
• 5. The parts are held together by sticky ends
  (preferably but not always)
• 6. Join permanently using DNA ligase (forms bonds
  between 5P and 3OH groups) forms covalent
  phosphodiester bonds
• 7. Introduce into bacteria
• 8. Plate and grow on antibiotic selection media
• 9. Select, analyze, etc.

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What else can one clone?
cDNA molecules
PCR-generated molecules (Polymerase chain
reaction)
Represent mRNA sequences
Represent mRNA or genomic DNA sequences
Figure 18.4
Figure 18.6
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• Scheme for Making DNA from RNA cDNA
• cDNA complementary DNA

Isolate poly A-tailed mRNAs, add poly A
primers REVERSE TRANSCRIPTASE SINGLE STRANDED
cDNA COMPLEMENTARY TO THE mRNAs DNA
POLYMERASE DOUBLE STRANDED cDNA CLONE AS USUAL
INTO YOUR PREFERRED CLONING VECTOR
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• Advantages for Using cDNAs
• 1. Represent actively expressed genes hence, not
  dormant genes
• 2. Represent processed information with no
  introns (spliced products)
• 3. No promoter sequences
• 4. No spacer DNA regions (stuff between genes)
• 5. Can be used almost directly to make proteins

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Building a DNA Library and Finding a DNA Clone of
Your Gene or Gene Product
Figure 18.7
Figure 18.8
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• THE ADVANTAGES OF A LIBRARY ARE
• 1. Amplification purposes, i.e. amplification of
  the entire genome, or all expressed genes
• 2. The cloning of distinct DNA or cDNA segments
• 3. A source for each specific part of the genome
  or all expressing genes
• 4. Representation of the entire genome or all
  expressing genes
• 5. Allow sophisticated, reproducible
  manipulations and searches

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MAKING A DNA LIBRARY The same
protocol for cloning a specific DNA or cDNA
sequence is used Except repeated (all at once)
many times to represent the genome, or all
expressing genes 1. Generate DNA fragments of
the target genome 2. Select different cloning
vectors -modified plasmids -bacterial viruses
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• 3. Libraries can be made to amplify cDNA or DNA
  fragments
• 4. Can use cDNA or genomic DNA directly
  (containing introns/exons, promoters, etc.)
• Libraries can be made to represent actively
  expressed genes, in other words, mRNAs or
  transcripts
• 6. Cloned cDNA or DNA fragments can be made to
  expressed protein products as well

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• How do you find a specific cDNA or genome
  segment?
• Build a cDNA or DNA library
• Then use Google or A Search Engine to find
  your cDNA or DNA clone
• In other words, some form of search information
  is required

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• The search information can be in several forms
• 1. As DNA sequences, short or long
• 2. As RNA sequences from transcripts
• Protein and/or protein sequences, via
• antibodies

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• WHAT ARE DNA OR RNA PROBES?
• Tagged, usually with radioactive nucleotides
• Single-stranded nucleic acid probes
• RNA or DNA forms
• Short or long sequences
• From any part of a gene

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Building a DNA Library and Finding a DNA Clone of
Your Gene or Gene Product
Figure 18.7
Figure 18.8
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• SEARCHING USING A DNA OR RNA PROBE

1
2
LABELLED DNA OR RNA PROBE
3
5
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• SEARCHING USING A PROTEIN PROBE (ANTIBODIES)

Proteins Made
1
ANTIBODY PROBE
2
Specific to the protein being searched
3
4
5
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• SEARCH FOR GENES IN REVERSE BY COMPLEMENTATION

MUTANT
1
2
3
4
5
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Genetic Engineering of Organisms

• Genetically
• Modified
• Organisms
• Biotechnology
• Gene Therapy

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Gene Therapy
Figure 19-20
Figure 19-21