More Molecular Genetic Technologies (Chapters 8, 9, 10) Polymerase Chain Reaction (PCR) Standard PCR method Real-time quantitative PCR DNA sequencing Manual dideoxy/automated fluorescent dye Pyrosequencing DNA Fingerprinting (DNA typing/profiling)
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Primers (anneal to flanking sequences)
How PCR works:
Begins with DNA containing a sequence to be amplified and a pair of synthetic oligonucleotide primers that flank the sequence.
Next, denature the DNA to single strands at 94˚C.
Rapidly cool the DNA (37-65˚C) and anneal primers to complementary single-straned sequences flanking the target DNA.
Extend primers at 70-75˚C using a heat-resistant DNA polymerase such as Taq polymerase derived from Thermus aquaticus.
Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1 million (220) to 35 trillion copies (245) of the target DNA.
Extend the primers at 70-75˚C once more to allow incomplete extension products in the reaction mixture to extend completely.
Cool to 4˚C and store or use amplified PCR product for analysis.
Hot water bacteria:
Taq DNA polymerase
Life at High Temperatures
by Thomas D. Brock
Biotechnology in Yellowstone
© 1994 Yellowstone Association for Natural Science
Anneal PCR Primers
Extend PCR Primers
Example thermal cycler protocol used in lab:
Step 17 min at 94˚CInitial Denature
Step 245 cycles of:
20 sec at 94˚CDenature
20 sec at 52˚CAnneal
1 min at 72˚CExtension
Step 37 min at 72˚CFinal Extension
Step 4Infinite hold at 4˚CStorage
Real-time Quantitative PCR:
Measures the abundance of DNA as it is amplified.
Useful for quantitatively measuring the levels of mRNA in a sample.
Uses reverse transcriptase to generate cDNA for the template.
Can also be used to quantitatively estimate fraction of DNA from various organisms in a heterogenous sample (e.g, can be used to measure abundance of different microbes in soil sample).
Fluorescent dye, SYBR Green, is incorporated into PCR reaction.
SYBR Green fluoresces strongly when bound to DNA, but emits little fluorescence when not bound to DNA.
SYBR Green fluorescence is proportional to the amount of DNA amplified, detected with a laser or other device.
Experimental samples are compared to control sample with known concentration of cDNA.
SYBR Green binds
to double-stranded DNA
Real-time Quantitative PCR amplification plot:
1980: Walter Gilbert (Biol. Labs) & Frederick Sanger (MRC Labs)
Dideoxy DNA sequencing (cont.):
Extension products in each of the four reaction mixtures also end with a different radio-labeled ddNTP (depending on the base).
Next, each reaction mixture is electrophoresed in a separate lane (4 lanes) at high voltage on a polyacrylamide gel.
Pattern of bands in each of the four lanes is visualized on X-ray film.
Location of “bands” in each of the four lanes indicate the size of the fragment terminating with a respective radio-labeled ddNTP.
DNA sequence is deduced from the pattern of bands in the 4 lanes.
Vigilant et al. 1989
Radio-labeled ddNTPs (4 rxns)
Sequence (5’ to 3’)
Automated Dye-Terminator dideoxy DNA Sequencing:
Original dideoxy DNA sequencing methods were time consuming, radioactive, and throughput was low, typically ~300 bp per run.
Automated DNA sequencing employs the same general procedure, but uses ddNTPs labeled with fluorescent dyes.
Combine 4 dyes in one reaction tube and electrophores in one lane on a capillary containing polyacrylamide.
UV laser detects dyes and reads the sequence.
Sequence data is displayed as colored peaks (chromatograms) that correspond to the position of each nucleotide in the sequence.
Throughput is high, up to 1,200 bp per reaction and 96 reactions every 3 hours with capillary sequencers.
Most automated DNA sequencers can load robotically and operate around the clock for weeks with minimal labor.
Applied Biosystems PRISM 377
(Gel, 34-96 lanes)
Applied Biosystems PRISM 3700
(Capillary, 96 capillaries)
Applied Biosystems PRISM 3100
(Capillary, 16 capillaries)
“virtual autorad” - real-time DNA sequence output from ABI 377
Trace files (dye signals) are analyzed and bases called to create chromatograms.
Chromatograms from opposite strands are reconciled with software to create double-stranded sequence data.
Fig. 8.11, Chromatogram of about 250 bp
Example showing how to read pyrosequencing
454 Life Sciences Genome Sequencer FLX
Generate 400 million nt in 10 hours
$5-7K USD per run
$1M for mammalian genome
DNA Fingerprinting (DNA typing/profiling)
DNA Fingerprinting (DNA typing/profiling)
Types of markers:
Repeated units of 5 to several 10 bp
Discovered by A. J. Jeffreys in 1985
Repeated units of 2-6 bp
Fig. 9.1 2nd edition, minisatellite repeat (VNTR)
Four criteria for selecting useful DNA fingerprinting markers:
(so that they are informative)
(so that they occur in only one location in the genome and there is no ambiguity about their number or position)
(so that the markers are independent)
(so that they are not correlated with selection or adaptation; unless selection of adaptation are to be studied; selection confounds estimates of population size parameters)
Microsatellites (short tandem repeats or STRs):
How to fingerprint alleged paternity using microsatellites:
Extract DNA from mother, baby, and alleged father.
Synthesize oligonucleotide microsatellite primers and label one primer with fluorescent dye (2 primers per microsatellite).
Amplify microsatellites using PCR from mother, baby, father.
Electrophores microsatellite PCR products on a DNA sequencer (w/polyacrylamide) with a flourescent size standard loaded in the same lane or capillary.
3-4 different microsatellites can be multiplexed in each lane or capillary by using 3-4 different fluorescent dyes.
Calculate size of each microsatellite relative to size standard (this size standard also can be run in the same gel lane or capillary using a 4th or 5th colored dye).
Sequence at least one copy of each allele to verify allele sizes.
Hypothetical gel pattern
for microsatellite heterozygous
for all individuals.
Paternity Analyses & Conclusions:
Single nucleotide polymorphisms (SNPs):
DNA sequences of most individuals are almost identical, >99%.
Single base pair differences occur about once every 500-1000 bp.
In most populations there is a common SNP, and several less common SNPs.
About 3 million SNPs occur in the human genome, and these are becoming popular genetic markers.
SNPs can be used just like other genotyping markers, but more loci typically must be used because only 4 alleles (G, G, C, T) are possible.
How to type SNPs:
DNA microarrays (DNA-chips, Gene-Chips, SNP-chips)
Fig. 8.14, Typing a SNP with an oligonucleotide.
How to type SNPs (cont.):
SNP chip is designed with an array of user defined oligonucleotides attached to the substrate (the SNP chip is the probe).
Oligonucleotides match each of the common and variant alleles in the population (all alleles of interest).
Target DNAs are labeled with a fluorescent tag and hybridized (or not) to the chip.
Fluorescence pattern is detected by a laser.
Because the oligonucleotides are known, the pattern indicates the type of alleles the individual possesses.
Many different alleles at thousands of different loci can be screened simultaneously in the same experiment.
Fig. 8.14b, SNP chip assay