VII. Molecular Biology
Techniques
Fall 2007
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.
Deoxyribonucleic Acid
Deoxyribonucleic Acid
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.
DNA Replication
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.
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.
Dot-Blot Hybridization
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Figure 1 DNA–DNA 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.
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.
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.
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.
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.
Southern Blot
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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.
Southern Blot
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]).
Northern Blot
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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.
Northern Blot
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.
Solution Hybridization
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.
Fluorescent In-Situ Hibridization
FISH
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.
How DNA Chips Are Made
• Used to examine DNA, RNA and other
substances
• Allow thousands of biological reactions to be
performed at once.
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.
Step 2: Manufacture substrate wafer.
• Companies use photolithography and other
nanomanufacturing techniques to turn glass and
plastic wafers into receptacles for the DNA
probes.
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.
DNA Chip
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
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.
Polymerase Chain Reaction
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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
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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.
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.
PCR
PCR
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.
TMA
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.
Ligase Chain Reaction
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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.
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.
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
References
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http://www.brc.dcs.gla.ac.uk/~drg/courses/bioinformatics_mscIT/slides/slides2/sld001.htm
•
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/G/GelBlotting.html
• http://www.bioteach.ubc.ca/MolecularBiology/IdentifyingDNA/
•
http://www.bio.davidson.edu/courses/genomics/Front/surfingenomics.html
• http://ccm.ucdavis.edu/cpl/Tech%20updates/TechUpdates.htm
• http://www.cdc.gov/ncidod/eid/vol7no2/pfaller.htm