VII. Molecular Biology Techniques

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VII. Molecular Biology Techniques

• Fall 2005

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Characteristics of Nucleic Acids

• Two types of nucleic acids RNA DNA
• DNA is encoded with four interchangeable
  "building blocks", called "bases", Adenine,
  Thymine, Cytosine, and Guanine, with Uracil
  rarely replacing Thymine
• RNA has five different bases adenine, guanine,
  cytosine, uracil, and more rarely thymine.

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Deoxyribonucleic Acid
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Deoxyribonucleic Acid
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DNA Replication

• Replication is performed by splitting (unzipping)
  the double strand down the middle via relatively
  trivial chemical reactions, and recreating the
  "other half" of each new single strand by
  drowning each half in a "soup" made of the four
  bases.
• Each of the "bases" can only combine with one
  other base, the base on the old strand dictates
  which base will be on the new strand.
• This way, each split half of the strand plus the
  bases it collects from the soup will ideally end
  up as a complete replica of the original, unless
  a mutation occurs.

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DNA Replication
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Nucleic Acid Probes

• Spontaneous pairing of complementary DNA strands
  forms basis for techniques used to detect and
  characterize genes.
• Probe technology used to identify individual
  genes or DNA sequences.
• Nucleic acid probe short strand of DNA or RNA of
  known sequence used to identify presence of
  complementary single strand of DNA in patient
  sample.
• Binding of 2 strands (probe and patient) known as
  hybridization.
• Two DNA strands must share at least 16 to 20
  consecutive bases of perfect complementarity to
  form stable hybrid.
• Match occurring as a result of chance less than 1
  in a billion.
• Probes labeled with marker radioisotope,
  fluorochrome, enzyme or chemiluminescent
  substrate.
• Hybridization can take place in solid support
  medium or liquid.

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Dot-Blot

• Dot-blot clinical sample applied to membrane,
  heated to denature DNA.
• Labeled probes added,
• Wash to remove unhybridized probe and measure
  reactants.
• Qualitative test only.
• May be difficult to interpret.

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Dot-Blot Hybridization

• Figure 1 DNADNA dot-blot hybridization between
  maize genomic DNA and a CaMV p-35S probe. Sample
  numbers coincide with those in ref. 1. Top row
  1, 100 transgenic 2, 10 transgenic 3, 5
  transgenic 4, 1 transgenic, 5, 0.5 transgenic
  6, historical maize negative control 7, water
  negative control 8, Diconsa sample K1. Bottom
  row 1, criollo sample B1 2, criollo sample B2
  3, criollo sample B3 4, criollo sample A1 5,
  criollo sample A2 6, criollo sample A3 7, Peru
  maize negative control P1 8, water negative
  control.

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Sandwich Hybridization

• Uses two probes, one bound to membrane and serves
  as capture target for sample DNA.
• Second probe anneals to different site on target
  DNA and has label for detection.
• Sample nucleic acid sandwiched between the two.
• Two hybridization events occur, increases
  specificity.
• Can be adapted to microtiter plates.

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Sandwich Hybridization

• Restriction endonucleases cleave both strands of
  double stranded DNA at specific sites,
  approximately 4 to 6 base pairs long.
• Further separated on the basis of size and charge
  by gel electrophoresis.
• Digested cellular DNA from patient/tissue added
  to wells in agarose gel and electric field
  applied, molecules move.
• Gel stained with ethidium bromid and vieuwed
  under UV light.

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Sandwich Hybridization

• Differences in restriction patterns referred to
  as restriction fragment length polymorphisms
  (RFLPs)
• Caused by variations in nucleotides within genes
  that change where the restriction enzymes cleave
  the DNA.
• When such a mutation occurs different size pieces
  of DNA are obtained.
• Caused by variations in nucleotides within genes
  that change where the restriction enzymes cleave
  the DNA.
• When such a mutation occurs different size pieces
  of DNA are obtained.

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Southern Blot

• DNA fragments separated by electrophoresis.
• Pieces denatured and transferred to membrane for
  hybridization reaction.
• Place membrane on top of gel and allow buffer
  plus DNA to wick up into it.
• Once DNA is on membrane heat or use UV ligth to
  crosslink strands onto membrane to immobilize.
• Add labeled probes for hybridization to take
  place.
• Probes added in excess so target molecules
  reanneal and more likely to attach to probe.

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Southern Blot

• The Southern Blot takes advantage of the fact
  that DNA fragments will stick to a nylon or
  nitrocellulose membrane. The membrane is laid on
  top of the agarose gel and absorbent material
  (e.g. paper towels or a sponge) is placed on top.
  With time, the DNA fragments will travel from the
  gel to the membrane by capillary action as
  surrounding liquid is drawn up to the absorbent
  material on top. After the transfer of DNA
  fragments has occurred, the membrane is washed,
  then the DNA fragments are permanently fixed to
  the membrane by heating or exposing it to UV
  light. The membrane is now a mirror image of the
  agarose gel.

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Southern Blot
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Southern Blot

• MOM blue, DAD yellow, and their four
  children D1 (the biological daughter), D2
  (step-daughter, child of Mom and her former
  husband red), S1 (biological son), and S2
  (adopted son,not biologically related his
  parents are light and dark green).

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Northern Blot

• Northern blots allow investigators to determine
  the molecular weight of an mRNA and to measure
  relative amounts of the mRNA present in different
  samples.
• RNA (either total RNA or just mRNA) is separated
  by gel electrophoresis, usually an agarose gel.
  Because there are so many different RNA molecules
  on the gel, it usually appears as a smear rather
  than discrete bands.
• The RNA is transferred to a sheet of special
  blotting paper called nitrocellulose, though
  other types of paper, or membranes, can be used.
  The RNA molecules retain the same pattern of
  separation they had on the gel.
• The blot is incubated with a probe which is
  single-stranded DNA. This probe will form base
  pairs with its complementary RNA sequence and
  bind to form a double-stranded RNA-DNA molecule.
  The probe cannot be seen but it is either
  radioactive or has an enzyme bound to it (e.g.
  alkaline phosphatase or horseradish peroxidase).
• The location of the probe is revealed by
  incubating it with a colorless substrate that the
  attached enzyme converts to a colored product
  that can be seen or gives off light which will
  expose X-ray film. If the probe was labeled with
  radioactivity, it can expose X-ray film directly.

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Northern Blot
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Solution Hybridization

• Both target nucleic acid and probe free to
  interact in solution.
• Hybridization of probe to target in solution is
  more sensitive than hybridization on solid
  support
• Requires less sample and is more sensitive.
• Probe must be single-stranded and incapable of
  self-annealing.
• Fairly adaptable to automation, especially tose
  using chemiluminescent labels.
• Assays performed in a few hours.

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Solution Hybridization
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In-Situ Hybridization

• Target nucleic acid found in intact cells.
• Provides information about presence of specific
  DNA targets and distribution in tissues.
• Probes must be small enough to reach nucleic
  acid.
• Radioactive or fluorescent tags used.
• Requires experience.

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Fluorescent In-Situ Hibridization FISH
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DNA Chip aka Microarrays

• A DNA chip (DNA microarray) is a biosensor which
  analyzes gene information from humans and
  bacteria.
• This utilizes the complementation of the four
  bases labeled A (adenine), T (thymine), G
  (guanine) and C (cytosine) in which A pairs with
  T and G pairs with C through hydrogen bonding.
• A solution of DNA sequences containing known
  genes called a DNA probe is placed on glass
  plates in microspots several microm in diameter
  arranged in multiple rows.
• Genes are extracted from samples such as blood,
  amplified and then reflected in the DNA chip,
  enabling characteristics such as the presence and
  mutation of genes in the test subject to be
  determined.
• As gene analysis advances, the field is gaining
  attention particularly in the clinical diagnosis
  of infectious disease, cancer and other maladies.
  

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How DNA Chips Are Made

• Used to examine DNA, RNA and other substances
• Allow thousands of biological reactions to be
  performed at once.

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Step 1 Make gene probes.

• Using conventional techniques such as polymerase
  chain reaction and biochemical synthesis, strands
  of identified DNA are made and purified. A
  variety of probes are available from commercial
  sources, many of which also offer custom
  production services.

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Step 2 Manufacture substrate wafer.

• Companies use photolithography and other
  nanomanufacturing techniques to turn glass and
  plastic wafers into receptacles for the DNA
  probes.

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Step 3 Deposit genetic sequences.

• Manufacturers use a variety of processes ranging
  from electrophoretic bonding to robotic
  deposition to adhere genetic material to the
  substrate. Cleanroom conditions and standards
  must be observed to attain the degree of
  contamination control needed during the
  deposition process.

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DNA Chip
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Drawbacks

• Stringency, or correct pairing, is affected by
• salt concentration
• Temperature
• concentration of destabilizing agent such as
  formamide or urea.
• If conditions not carefully controlled mismatches
  can occur.
• Patient nucleic acid may be present in small
  amounts, below threshold for probe detection.
• Sensitivity can be increased by amplification
  target, probe and signal

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Target Amplification

• In-vitro systems for enzymatic replication of
  target molecule to detectable levels.
• Allows target to be identified and further
  characterized.
• Examples Polymerase chain reaction,
  transcription mediated amplification,, strand
  displacement amplification and nucleic acid
  sequence-based amplification.

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Polymerase Chain Reaction

• Capable of amplifying tiny quantities of nucleic
  acid.
• Cells separated and lysed.
• Double stranded DNA separated into single
  strands.
• Primers, small segments of DNA no more than 20-30
  nucleotides long added.
• Primers are complementary to segments of opposite
  strands of that flank the target sequence.
• Only the segments of target DNA between the
  primers will be replicated.
• Each cycle of PCR consists of three cycles
• denaturation of target DNA to separate 2 strands.
• annealing step in which the reaction mix is
  cooled to allow primers to anneal to target
  sequence
• Extension reaction in which primers initiate DNA
  synthesis using a DNA polymerase.
• These three steps constitute a thermal cycle
• Each PCR cycle results in a doubling of target
  sequences and typically allowed to run through 30
  cycles, one cylce takes approximately 60-90
  seconds.

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Taq

• Taq polymerase ("Taq pol") is a thermostable
  polymerase isolated from thermus aquaticus, a
  bacterium that lives in hot springs and
  hydrothermal vents.
• "Taq polymerase" is an abbreviation of Thermus
  Aquaticus Polymerase.
• It is often used in polymerase chain reaction,
  since it is reasonably cheap and it can survive
  PCR conditions.

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PCR
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PCR
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Transcription Mediated Amplification

• TMA is the next generation of nucleic acid
  amplification technology.
• TMA is an RNA transcription amplification system
  using two enzymes to drive the reaction RNA
  polymerase and reverse transcriptase.
• TMA is isothermal the entire reaction is
  performed at the same temperature in a water bath
  or heat block. This is in contrast to other
  amplification reactions such as PCR or LCR that
  require a thermal cycler instrument to rapidly
  change the temperature to drive the reaction.
• TMA can amplify either DNA or RNA, and produces
  RNA amplicon, in contrast to most other nucleic
  acid amplification methods that only produce DNA.
  
• TMA has very rapid kinetics resulting in a
  billion fold amplification within 15-30 minutes.

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TMA
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QB Replicase

• Uses an RNA directed RNA polymerase that
  replicates the genomic RNA of a bacteriophage
  named QB.
• The RNA genome of QB is essentially the only
  substrate recognized by the polymerase.
• Because a short probe can be inserted into the QB
  RNA this becomes the system for amplification.
• After the probe has annealed to the target,
  unbound probe is treated with RNase and washed
  away.
• The hybridized probe is RNase resistant.
• When QB replicase is added the probe is
  enzymatically replicated to detectable levels.

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Ligase Chain Reaction

• The LCR test employs four synthetic
  oligonucleotide probes to anneal at specific
  target sites on the cryptic plasmid.
• Each pair of probes hybridize close together on
  the target DNA template.
• Once the probes are annealed, the gap is filled
  by DNA polymerase and close by the ligase enzyme.
• This two-step process of closing the gap between
  annealed probes makes the LCR, in theory, more
  specific than PCR technology.
• The ligated probe pairs anneal to each other and,
  upon denaturation, form the template for
  successive reaction cycles, thus producing a
  logarithmic amplification of the target sequence.
  
• Like PCR, LCR is made in a thermocycler.
• The LCR product is detected in an automated
  instrument that uses an immunocolorimetric bead
  capture system.
• At the end of the LCR assay, amplified products
  are inactivated by the automatic addition of a
  chelated metal complex and a oxidizing agent.

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Drawbacks of Amplification Systems

• Potential for false-positive results due to
  contaminating nucleic acids.
• PCR and LCR, DNA products main source of
  contamination.
• QB replicase and TMA, RNA products are possible
  contaminants.
• Must have product inactivation as part of QC
  program.
• Separate preparation areas from amplification
  areas and use of inactivation systems such as UV
  light help alleviate contamination.
• Very expensive.
• Closed system, automation will also decrease
  number of problems.

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Future of Molecular Diagnostic Techniques

• Despite expense may be times that rapid diagnosis
  will result in decreased cost.
• Example Mycobacteria - quick diagnosis no need
  for expensive respiratory isolation.
• Detection of multi-drug resistant M. Tuberculosis
  will lead to more timely public health measures.
• Incredibly useful in serology and microbiology.
• Increased specificity and sensitivity of
  molecular testing will become the standard of
  practice in immunology and microbiology.
• Testing will continue to become more rapid as
  assays are automated which will also bring down
  the costs.
• Author states will not replace culture for
  routine organisms, but it already is, and as DNA
  chip technology improves, the ability to test for
  multiple organisms will become easier

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Signal Amplification

• Replicates signal rather than either the target
  or the probe.
• Based on the reporter group (the labeled tag)
  being attached in greater numbers to the probe
  molecule or increasing the intensity generated by
  each labeled tag.
• Patient nucleic acid not replicated or amplified
  technique is less prone to contamination.
• Sensitivity is lower.
• Branched chain signal amplification employs
  several simultaneous hybridization steps.
• Author states similar to decorating a Christmas
  tree and involves several sandwich
  hybridizations.
• first, target specific oligonucleotide probes
  captures target sequence to solid support.
• Second set of target specific probes called
  extenders hybridize to adjoining sequences and
  act as binding site for large piece called
  branched amplification multimer.
• Each branch has multiple side branches capable of
  binding numerous oligonucleotides.
• Branched chains are well suited to detection of
  nucleic acid targets with sequence heterogeneity
  such as hepatitis C and HIV.

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References

• http//www.brc.dcs.gla.ac.uk/drg/courses/bioinfor
  matics_mscIT/slides/slides2/sld001.htm
• http//users.rcn.com/jkimball.ma.ultranet/BiologyP
  ages/G/GelBlotting.html
• http//www.bioteach.ubc.ca/MolecularBiology/Identi
  fyingDNA/
• http//www.bio.davidson.edu/courses/genomics/Front
  /surfingenomics.html
• http//ccm.ucdavis.edu/cpl/Tech20updates/TechUpda
  tes.htm
• http//www.cdc.gov/ncidod/eid/vol7no2/pfaller.htm