Methods in Biomedical Science BMSC 416: Lecture 1

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Methods in Biomedical Science (BMSC 416)
Lecture 1

• Course Overview, DNA Cloning, PCR, Southern Blot,
  Northern Blot

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Course Directors

• David Keating, Assistant Professor of
  Microbiology and Immunology (Maguire, Rm 3931)
• Nancy Zeleznik-Le Associate Professor of Medicine
  (Cancer Center Room 337)

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Course Objectives

• The objective of this course is to familiarize
  first year graduate students with methods that
  are used in biomedical research.
• Techniques covered including molecular biology,
  tissue culture, transgenic model systems,
  imaging, biochemistry, electrophysiology, and
  immunology (some new, some not).  

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Rationale

• The theory behind the techniques, as well as
  practical considerations will be discussed.
• The course will provide students with a
  conceptual foundation for the critical evaluation
  of published experiments, and guide the choice of
  methods used in their own research.

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Lecturers

• Lectures will be given by faculty with practical
  and theoretical knowledge of the methods
  discussed during any particular lecture.
• 10 faculty will participate.
• Despite the large number of lecturers, we will
  strive to provide continuity within the course.

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Resources

• Alberts et al., Molecular Biology of the Cell
  Fourth edition, 2002
• Boron and Boulpaep, Medical Physiology, First
  Edition, 2003
• Katzung, Basic and Clinical Pharmacology, Seventh
  Edition, 1998
• Wilson and Hunt, Molecular Biology of the Cell, A
  Problems Approach, Fourth Edition, 2002).

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Resources

• For methods that are not described in these text
  books, appropriate reading materials will be
  provided to the students prior to each lecture.

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Grading

• Students will be graded by three examinations
• Exam 1 Monday, August 20
• Exam 2 Monday, September 17
• Exam 3 Monday, October 1
• Each exam will be preceded by a problem solving
  session (chance to ask questions).
• Completion of problem sets will contribute 25 to
  your final grade (turn in at beginning of problem
  solving session-photocopy!).

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Problem Sets and Lecture Notes

• Graduate Website http//library.luhs.org/grad/mai
  n.htm
• (Also can enter through LUHS Library)
• Calendar, Problem Sets, Class Notes
• http//library.luhs.org/grad/07august.htm
• (you may not be able to access from home)

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Questions?????
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Lecture 1 Molecular Biology Methods

• Techniques covered in this lecture
• Brief Overview of DNA Cloning
• PCR
• Northern Blot
• Southern Blot
• Reading Alberts 491-504, 508-509 

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Learning Objectives

• DNA Cloning
• Be able to define gene cloning, and the reasons
  for its use.
• PCR
• Be able to clearly articulate the steps and
  enzymes and reagents involved in PCR.
• Explain why PCR yields such a tremendous
  amplification of DNA, as well as technical
  concerns that can limit its effectiveness.
• Be able to describe alternative uses of PCR such
  as RTPCR and colony PCR,
• Southern Blot
• Explain the steps involved in a Southern Blot.
• Be able to define hybridization, probe, and
  stringency.
• Describe the advantages and limitations of
  Southern Blots.
• Be able to articulate the situations in which a
  Southern Blot is more useful than PCR.
• Northern Blot
• Be able to describe the differences between RNA
  and DNA that are relevant to hybridization
  experiments.
• Be able to articulate the differences in
  procedure between a Northern and a Southern Blot,
  and why they are necessary.
• Define a quantitative Northern Blot, and what
  additional steps are necessary to to perform a
  quantitative Northern Blot.
• Under what conditions are techniques such as
  RTPCR or realtime PCR used instead of Northern
  Blots.
• Are microarrays a Northern Blot or a Southern
  Blot?

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What is DNA cloning?

• Insertion of DNA into self-replicating element.

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What is the purpose of DNA cloning?

• Cloning allows amplification of the DNA of
  interest.

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Overview of Cloning
Target DNA
Vector
(Plasmid)
In vitro
(Ligation)
Introduce into suitable host strain
(Transformation or electroporation not
transfection )
In vivo
Identification of clones of interest
More information, Alberts 491-504
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Why can we do with cloned genes?

• To determine their primary sequence
• Expression of the gene or gene product
• Manipulation of the gene or gene product
• Mutagenesis
• Tagging of protein product
• Measurement of transcription and translation

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PCR
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Overview of Cloning
How do you generate the target DNA?
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Identification of genes in a heterogeneous
population

• The problem how do you identify and selectively
  amplify a single gene in a heterogeneous
  population?
• Traditional method make restriction maps, clone
  restriction fragments, a subset of which would be
  expected to contain the gene of interest, and
  screen for the correct clone.
• This approach can be very labor intensive and
  could take weeks/years to accomplish.

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The Polymerase Chain Reaction (PCR)

• An alternative method was developed in the 1980s
  (Kary Mullis) that allowed the selective
  amplification of any piece of DNA in a
  heterogeneous population-in less than 4 hrs!
• The only requirement is that you know or can
  infer the sequence of your gene of interest.
• Furthermore, this technology has been co-opted to
  produce extraordinary advancements in a variety
  of fields of study.

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Requirements for a PCR reaction

• Template (DNA, cDNA, whole cells)
• dNTPs (dATP, dCTP, dGTP, dTTP)
• Thermostable polymerase
• Primers oligonucleotides that are complimentary
  to your gene of interest, and prime DNA synthesis
  (5-3) towards each other

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The PCR Reaction
5-GCATCCCGGGATAGCTAGTGACTAGC-3 3-CGTAGGGCCCTATC
GATCACTGATCG-5
Template DNA
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The PCR Reaction
5-GCATCCC-3
3-CGTAGGGCCCTATCGATCACTGATCG-5
5-GCATCCCGGGATAGCTAGTGACTAGC-3
3-CTGATCG-5
In vitro DNA synthesis
5-GCATCCCGGGATAGCTAG.-3
3-CGTAGGGCCCTATCGATCACTGATCG-5
5-GCATCCCGGGATAGCTAGTGACTAGC-3
3-..CCCTATCGATCACTGATCG-5
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Results from one round of DNA synthesis
5-GCATCCCGGGATAGCTAGTGACTAGC-3 3-CGTAGGGCCCTATC
GATCACTGATCG-5
Before
5-GCATCCC-3
5-GCATCCCGGGATAGCTAGTGACTAGC-3
3-CGTAGGGCCCTATCGATCACTGATCG-5
After
5-GCATCCCGGGATAGCTAGTGACTAGC-3
3-CGTAGGGCCCTATCGATCACTGATCG-5
3-CTGATCG-5
What happens if we repeat the cycle?
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Overview of PCR
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Overview of PCR

• Each cycle of PCR results in an exponential
  increase in DNA.
• 35-cycles will produce 34,359,738,370 copies!
• For a small gene (less than 1kb), this can be
  carried out in a little over an hour!

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Types of template DNA for PCR

• Purified DNA or cDNA PCR operates most
  efficiently when purified DNA is used as a
  template.
• Whole cells Although less efficient, PCR can be
  very effective.
• Environmental samples.

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Uses of PCR

• Most applications involving DNA require
  relatively large quantities.
• Because PCR can generate a tremendous
  amplification of DNA, it facilitates many DNA
  manipulations.

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Uses of PCR

• Cloning amplification of DNA for cloning genes,
  bypassing the need for restriction enzymes and
  electrophoresis.
• Examination of gene expression cDNA prepared
  from mRNA can be used as a template, thus
  allowing measurement of gene expression.
• Diagnostic PCR allows specific amplification of
  a gene of interest from a very dilute sample.
  However, if the gene is not present in the
  sample, it cannot be amplified. Therefore, PCR
  can be an excellent diagnostic.

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Examination of gene expression RT-PCR
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Real Time PCR
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Uses of PCR

• Cloning amplification of DNA for cloning genes,
  bypassing the need for restriction enzymes and
  electrophoresis.
• Examination of gene expression cDNA prepared
  from mRNA can be used as a template, thus
  allowing measurement of gene expression.
• Diagnostic PCR allows specific amplification of
  a gene of interest from a very dilute sample.
  However, if the gene is not present in the
  sample, it cannot be amplified. Therefore, PCR
  can be an excellent diagnostic.

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Diagnostic PCR Colony PCR

Add bacterial cells to PCR reaction
PCR Reaction
Colony does not contain plasmid with insert of
interest
Colony Contains plasmid with insert of interest
Agarose Gel
Agarose Gel
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Specificity and contamination

• Purity of samples the extraordinary power of PCR
  means that contaminants can also be amplified.
• Primer choice if primers anneal to the wrong
  genomic region, this region will be amplified.
• Temperature Specific base pairing of primers to
  template DNA only occurs in a narrow range of
  temperature and salt concentration.

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Technical Considerations

• Polymerase fidelity the thermostable Taq
  polymerase is commonly used for PCR. However, it
  incorporates the incorrect base at a rate of
  0.0002-0.001 errors/bp.
• The so called proof-reading polymerases (e.g.
  PFU polymerase) result in a lower error rate (1.6
  x 10-6 errors/base), but are often more difficult
  to use.
• The choice of polymerase depends on the required
  outcome (e.g. gene cloning vs analytical
  experiment).

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Technical Considerations

• Length of amplified sequence. PCR works best for
  shorter sequences (lt5Kb).
• Longer sequences can be amplified, but require
  the use of alternative protocols (e. g. mixed
  polymerases).

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Final Thought

• PCR is an unbelievably powerful technique.
  However, the ability to amplify DNA means that
  potentially one contaminating molecule of DNA can
  result in yield anomalous results.
• Be careful!

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Southern Blots
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Overview of Cloning
How do you Identify the target DNA?
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How do you identify the correct fragment?

• PCR
• However, PCR is ineffective for large genes or
  genes of undetermined nucleotide sequence.
• Southern blots allow identification of genes of
  any size, without prior knowledge of nucleotide
  sequence.

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Southern Blot Requires Hybridization
5-GCATCCCGGGATAGCTAGTGACTAGC-3 3-CGTAGGGCCCTATC
GATCACTGATCG-5
Template DNA
Thus, labeled probes can be used to detect the
presence of a specific DNA in a complex
population
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Overview of Southern Blot
Target DNA
Detect by autoradiography
Digest with Restriction Enzymes
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis
Denature DNA
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Overview of Southern Blot
Target DNA
Detect by autoradiography
Digest with Restriction Enzymes
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis
Denature DNA
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Some restriction enzymes produce Blunt Ends
Restriction enzymes that cut within the center of
the recognition sequence produce blunt ends.
5-GCATCCCGGGATAGCTAGTGACTAGC-3 3-CGTAGGGCCCTATC
GATCACTGATCG-5
5-GCATCCC GGGATAGCTAGTGACTAGC-3 3
-CGTAGGG
CCCTATCGATCACTGATCG-5
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Some restriction enzymes produce Sticky Ends

• Restriction enzymes that make staggered cuts
  within the recognition sequence produce cohesive
  or sticky ends.

5-GCATGAATTCATAGCTAGTGACTAGC-3 3-CGTACTTAAGTATC
GATCACTGATCG-5
5-GCATGA ATTCATAGCTAGTGACTAGC-3
3-CGTACTTA AGTATCGATCACTGATCG-5
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Overview of Southern Blot
Target DNA
Detect by autoradiography
Digest with Restriction Enzymes
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis
Denature DNA
Restriction enzyme digestion and fractionation
same as discussed previously.
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Gel Electrophoresis

• Small fragments (lt500 bp) can be fractionated by
  electrophoresis on polyacrylamide gels.
• Most fractionation of DNA is done by agarose
  gels.
• Advantages inexpensive, simple to prepare,
  allows fractionation of large fragments (507 Da
  /ATP).

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Overview of Southern Blot
Target DNA
Detect by autoradiography
Digest with Restriction Enzymes
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis
Denature DNA
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Denaturation of DNA

• Detection of a specific DNA sequence by
  hybridization requires separation of double
  strand DNA.
• DNA can be denatured by either temperature or pH.
  
• pH is used for southern blots (high temps would
  melt the agarose gel!).

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Overview of Southern Blot
Target DNA
Detect by autoradiography
Digest with Restriction Enzymes
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis
Denature DNA
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Transfer of DNA to Solid Support

• The key aspect to all blots (southern, northern,
  western) is the transfer of the fractionated
  nucleic acid (or protein in the case of a western
  blot) to a solid support-a process called
  blotting.

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Blotting of DNA
(nitrocellulose is also used)
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Blotting of DNA

• Once the material has been transferred to the
  solid support, it is then permanently fixed to
  the membrane by either heat or treatment with UV
  light.

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Overview of Southern Blot
Target DNA
Detect by autoradiography
Digest with Restriction Enzymes
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis
Denature DNA
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Hybridization
Radiolabeled Probe
32PO4- 5-GCATCCC-3
3-CGTAGGGCCCTATCGATCACTGATCG-5
Template DNA
Mix probe and DNA at high temp, allow to cool
32PO4-5-GCATCCC-3
3-CGTAGGGCCCTATCGATCACTGATCG-5
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Overview of Southern Blot
Detect by autoradiography
Target DNA
Digest with Restriction Enzymes
Wash
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis
Denature DNA
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Hybridization

• Membrane is incubated in the presence of the
  hybridization solution a process called
  pre-hybridization)
• Probe is then boiled (to convert to single
  stranded form), and then added to incubation mix.

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Hybridization and Washing

• Blot is then washed with solutions of increasing
  temperature and/or decreased salt until label
  bound nonspecfically to membrane is reduced.

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Overview of Southern Blot
Target DNA
Detection
Digest with Restriction Enzymes
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis
Denature DNA
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Detection of Hybridized DNA

• Radioactivity bound to the membrane can be
  detected by autoradiography (in which the
  radioactivity is exposed to X-ray film.
• Alternatively, radioactivity can be detected by
  phosphorimaging, which uses a matrix which will
  can be induced to emit light upon exposure to
  radioactivity.
• Increasingly, non-radioactive methods are used to
  visualize hybridization.

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Example of a Southern Blot
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Theoretical concerns

• Hybridization is a competition between specific
  and non-specific binding. Reduction in salt or
  increase in temperature favor specific binding.
• The appropriate hybridization/washing conditions
  (often called stringency) often have to be
  empirically determined.

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Uses of Southern blots

• Mapping of genes (RFLP)
• Identification of clones that harbor a gene of
  interest.
• Identification of large scale genomic
  rearrangments
• Qualitative genomic comparison in multiple
  species (zoo blots).

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Disadvantages of Southern blots

• Southerns are time consuming, requiring two days
  or more to complete.
• Southerns require specialized equipment.

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Are southern hybridizations still valuable in the
era of the sequenced genome?

• The advantages of Southern Blots will prevent
  them from completely disappearing anytime soon.
• The microarray technology widely used today is a
  modified Southern blot.

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Northern Blots
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Northern Blots

• A method to identify a specific RNA transcript in
  a diverse population.

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Major differences between studying DNA and RNA

• Genomic DNA is composed of a relatively small
  number of large macromolecule, while RNA is a
  collection of smaller macromolecules.
• RNA is single stranded, yet adopts a folded
  conformation in the absence of a denaturant.
• RNA is very sensitive to alkaline pH and
  nucleases (many of which are present on human
  skin).

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History of Northern Blots

• The history of the Northern blot mirrors the
  history of the Southern Blot.
• Because of the challenges associated with RNA
  (instability, nucleases, secondary structure), it
  took several changes in protocol to make the
  method successful.

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History of Northern blots

• The first description of a gel transfer/hybridizat
  ion method for RNA was made by Alwine et al, who
  as a joke called it a Northern Blot.

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Overview of Northern Blot
Detection
Extract target RNA
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis under denaturing
conditions
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Overview of Northern Blot
Detection
Extract Target RNA
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis under denaturing
conditions
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Purification of RNA

• RNA overall has similar properties to DNA, so the
  same types of purification schemes are used.
• However, RNA is very sensitive to alkaline pH.

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Overview of Northern Blot
Detection
Extract Target RNA
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis under denaturing
conditions
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Electophoretic fractionation of RNA

• RNA can adopt a folded conformation that would
  prevent hybridization.
• Furthermore, alkaline conditions can not be used
  to produce single stranded RNA.
• Therefore the fractionation is performed under
  conditions that prevent secondary structure of
  RNA.

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Electrophoretic fractionation of RNA

• Formaldehyde is the most common denaturant,
  although urea is increasingly used.
• The electrophoresis is generally carried out on
  agarose, although acrylamide can be used in some
  instances.

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Overview of Northern Blot
Detection
Extract Target RNA
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis under denaturing
conditions
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Transfer of RNA to a solid support

• The transfer of RNA is carried out under the same
  conditions as transfer of DNA except the alkaline
  denaturation step is unnecessary.
• The RNA is stable once bound to the solid support.

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Overview of Northern Blot
Detection
Extract Target RNA
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis under denaturing
conditions
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Preparation of labeled probe

• RNA can in principle be used a probe (providing
  it is complementary to the RNA of interest).
• However, DNA is the most common probe.
• DNA probes are prepared from either the genomic
  DNA or from cDNA.

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Overview of Northern Blot
Detection
Extract Target RNA
Add labeled probe
Transfer to solid support
Fractionate by electrophoresis under denaturing
conditions
82
Hybridization and washing of Northern blots

• Hybridization of Northern blots is essentially
  the same as Southern blots.
• Washing is also carried out by the same method.

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Detection of hybridized probe

• Hybridization is generally carried out by
  autoradiography with X-ray film or
  phosphorimaging.
• Non-radioactive methods are less common in
  Northern blots than they are in Southern blots,
  but are available.

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Theoretical concerns

• The issues of specific vs non-specific
  hybridization are largely the same for a Northern
  blot as they are for a Southern.
• The RNA-DNA hybrid may be less stable than some
  DNA-DNA hybridizations, so experiment has to
  sometimes be carried out at lower stringencies.

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Northern blots can be used to quantify mRNA levels

• Northerns can be used to quantify the relative
  expression of a gene under different conditions,
  in different strains, or genetic backgrounds.
• However, this requires the use of strict
  controls.

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Requirements for quantitative Northerns

• The total amount of RNA must be the same between
  samples (easily assayed spectrophotometrically)
• In addition, it is essential to insure that the
  total amount of RNA transferred to the solid
  support is the same.

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Requirements for quantitative Northerns

• The most common method to insure that the amount
  of transferred RNA is the same is to remove the
  labeled probe from the gel (stripping).
• A new probe of a constitutively expressed gene
  (such as actin or GAPDH) is then used as a probe.
  
• The actin expression is then used to control for
  differences in RNA transfer etc.

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Example of a Northern Blot

•  

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Advantages of Northern blots

• Northern blots are very easy to quantify, and can
  in principle be used to determine an absolute
  level of expression of a given gene.
• Northern blots can be very informative regarding
  the size of transcript, which can then be used to
  identify introns and operons.

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Disadvantages of Northern blots 

• Like Southern blots, Northerns are somewhat
  laborious and require 2-3 days to complete.
• The larger issue is sensitivity for many genes
  the Northern blot is not sensitive enough to
  allow detection of poorly transcribed genes or
  unstable transcripts.
• As a result, methods such as RT PCR are and real
  time PCR, and microarrays are more successful.

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Final Thought

• Blots are not as commonly used as they used to
  be, but are the backbone of the array techniques.
• In fact the primary microarray patent was
  determined to belong to Southern, because it is
  essentially the same technique.
• The Northern blot is likely going to remain an
  important tool due to its ability to generate
  information about the size and number of
  transcripts which are difficult by other
  techniques.