DNA SEQUENCING AND TYPES OF DNA SEQUENCING.ppt

DNA sequencing

1. Molecular Genetic Methods - Lecture Topics Chapter
1.Polymerase Chain Reaction (PCR) 7
2.DNA sequencing (manual/automated) 7
3.DNA Fingerprinting (DNA typing/profiling) 8
4.Single nucleotide polymorphisms (SNPs) 9

2. Practical applications of PCR, Sequencing, Fingerprinting, & SNPs:
Amplify DNA for Cloning (PCR)
Amplify DNA for sequencing without cloning (PCR)
DNA sequencing reaction (PCR)
Mapping genes and regulatory sequences
Linkage analysis (identify genes for traits/diseases)
Diagnose disease
Pathogen screening
Sex determination
Forensic analysis
Paternity/maternity (relatedness)
Behavioral ecology studies (relatedness)
Molecular systematics and evolution (comparing homologous
sequences in different organisms)
Population genetics (theoretical and applied)
Physiological genetics (studying basis of adaptation)
Livestock pedigrees (optimize breeding)
Wildlife management (stock identification/assessment)
Detection of Genetically Modified Food (GMOs)

3. Background on the Polymerase Chain Reaction (PCR)
Ability to generate identical high copy number DNAs made possible
in the 1970s by recombinant DNA technology (i.e., cloning).
Cloning DNA is time consuming and expensive (>>$15/sample).
 Probing libraries can be like hunting for a needle in a haystack.
PCR, “discovered” in 1983 by Kary Mullis, enables the amplification
(or duplication) of millions of copies of any DNA sequence with
known flanking sequences.
Requires only simple, inexpensive ingredients and a couple hours.
DNA template
Primers (anneal to flanking sequences)
DNA polymerase
dNTPs
Mg
2+
Buffer
Can be performed by hand or in a machine called a thermal cycler.
1993: Nobel Prize for Chemistry

4. How PCR works:
1.Begins with DNA containing a sequence to be amplified and a pair
of synthetic oligonucleotide primers that flank the sequence.
2.Next, denature the DNA to single strands at 94˚C.
3.Rapidly cool the DNA (37-65˚C) and anneal primers to
complementary s.s. sequences flanking the target DNA.
4.Extend primers at 70-75˚C using a heat-resistant DNA
polymerase such as Taq polymerase derived from Thermus
aquaticus.
5.Repeat the cycle of denaturing, annealing, and extension 20-45
times to produce 1 million (2
20
)to 35 trillion copies (2
45
) of the
target DNA.
6.Extend the primers at 70-75˚C once more to allow incomplete
extension products in the reaction mixture to extend completely.
7. Cool to 4˚C and store or use amplified PCR product for analysis.

5. Hot water bacteria:
Thermus aquaticus
Taq DNA polymerase
Life at High Temperatures
by Thomas D. Brock
Biotechnology in Yellowstone
© 1994 Yellowstone Association for Natural Science
http://www.bact.wisc.edu/Bact303/b27

6. Fig. 7.23
Denature
Anneal PCR Primers
Extend PCR Primers
w/Taq
Repeat…

7. Example thermal cycler protocol used in lab:
Step 1 7 min at 94˚C Initial Denature
Step 2 45 cycles of:
20 sec at 94˚C Denature
20 sec at 52˚C Anneal
1 min at 72˚C Extension
Step 3 7 min at 72˚C Final Extension
Step 4 Infinite hold at 4˚C Storage

8. DNA Sequencing
DNA sequencing = determining the nucleotide sequence of DNA.
Developed by Frederick Sanger in the 1970s.
1980: Walter Gilbert (Biol. Labs) & Frederick Sanger (MRC Labs)

9. Manual Dideoxy DNA sequencing-How it works:
1.DNA template is denatured to single strands.
2.DNA primer (with 3’ end near sequence of interest) is annealed to the
template DNA and extended with DNA polymerase.
3.Four reactions are set up, each containing:
1.DNA template
2.Primer annealed to template DNA
3.DNA polymerase
4.dNTPS (dATP, dTTP, dCTP, and dGTP)
4.Next, a different radio-labeled dideoxynucleotide (ddATP, ddTTP,
ddCTP, or ddGTP) is added to each of the four reaction tubes at
1/100th the concentration of normal dNTPs.
5.ddNTPs possess a 3’-H instead of 3’-OH, compete in the reaction with
normal dNTPS, and produce no phosphodiester bond.
6.Whenever the radio-labeled ddNTPs are incorporated in the chain,
DNA synthesis terminates.
7.Each of the four reaction mixtures produces a population of DNA
molecules with DNA chains terminating at all possible positions.

10. Manual Dideoxy DNA sequencing-How it works (cont.):
8.Extension products in each of the four reaction mixtures also end
with a different radio-labeled ddNTP (depending on the base).
9.Next, each reaction mixture is electrophoresed in a separate lane (4
lanes) at high voltage on a polyacrylamide gel.
10.Pattern of bands in each of the four lanes is visualized on X-ray film.
11.Location of “bands” in each of the four lanes indicate the size of the
fragment terminating with a respective radio-labeled ddNTP.
12.DNA sequence is deduced from the pattern of bands in the 4 lanes.

11. Fig. 7.20
Vigilant et al. 1989
PNAS 86:9350-9354

12. Short products
Long products
Radio-labeled ddNTPs (4 rxns)
Sequence (5’ to 3’)
G
G
A
T
A
T
A
A
C
C
C
C
T
G
T

13. Automated Dye-Terminator DNA Sequencing:
1.Dideoxy DNA sequencing was time consuming, radioactive, and
throughput was low, typically ~300 bp per run.
2.Automated DNA sequencing employs the same general procedure,
but uses ddNTPs labeled with fluorescent dyes.
3.Combine 4 dyes in one reaction tube and electrophores in one lane on
a polyacrylamide gel or capillary containing polyacrylamide.
4.UV laser detects dyes and reads the sequence.
5.Sequence data is displayed as colored peaks (chromatograms) that
correspond to the position of each nucleotide in the sequence.
6.Throughput is high, up to 1,200 bp per reaction and 96 reactions
every 3 hours with capillary sequencers.
7.Most automated DNA sequencers can load robotically and operate
around the clock for weeks with minimal labor.

14. Applied Biosystems PRISM 377
(Gel, 34-96 lanes)
Applied Biosystems PRISM 3100
(Capillary, 16 capillaries)
Applied Biosystems PRISM 3700
(Capillary, 96 capillaries)

15. “virtual autorad” - real-time DNA sequence output from ABI 377
1.Trace files (dye signals) are analyzed and bases
called to create chromatograms .
2.Chromatograms from opposite strands are
reconciled with software to create double-
stranded sequence data.

16. DNA Fingerprinting (DNA typing/profiling)
No two individuals produced by sexually reproducing organisms
(except identical twins) have exactly the same genotype.
Why?
Crossing-over of chromosomes in meiosis prophase I.
Random alignment of maternal/paternal chromosomes in
meiosis metaphase I.
Mutation
DNA replication errors (same effect as mutation)

17. DNA Fingerprinting (DNA typing/profiling)
Types of markers:
RFLPs (restriction sites)
Length polymorphism detected by PCR
Allele specific oligonucleotide probes
Repeated DNA
Minisatellites (VNTRs = variable number tandem repeats)
Repeated units of 5 to several 10 bp
Discovered by A. J. Jeffreys in 1985
Microsatellites (STRs = short tandem repeats)
Repeated units of 2-6 bp
5’-TAATAATAATAATAATAA-3’
3’-ATTATTATTATTATTATT-5’

18. Fig. 9.1, minisatellite repeat (VNTR)

19. Four criteria for selecting useful DNA fingerprinting markers:
1.Markers should be polymorphic.
(so that they are informative)
2.Markers should be single locus.
(so that they occur in only one location in the genome and there
is no ambiguity about their number or position)
3.Markers should be neutral.
(so that they are not correlated with selection or adaptation;
unless selection of adaptation are to be studied)
4.Markers should be located on different chromosomes .
(so that the markers are independent)

20. Microsatellites (short tandem repeats):
Heterozygote Male 5’-TAATAATAATAATAATAATAA----3’
Female5’-TAATAATAATAATAATAATAATAA-3’
Homozygote Male 5’-TAATAATAATAATAA-3’
(different allele) Female5’-TAATAATAATAATAA-3’
One proposed explanation for their fast rate of evolution is
slippage during DNA replication.
Excellent marker for DNA fingerprinting because:
1.Polymorphic (fast-evolving)
1.Single locus
1.Neutral (non-coding)
1.Common throughout genomes of most organisms

21. How to fingerprint alleged paternity using microsatellites:
1.Extract DNA from mother, baby, and alleged father.
2.Synthesize oligonucleotide microsatellite primers and label one
primer with fluorescent dye (2 primers per microsatellite).
3.Amplify microsatellites using PCR from mother, baby, father.
4.Electrophores microsatellite PCR products on a DNA sequencer
(w/polyacrylamide) with a flourescent size standard loaded in
the same lane or capillary.
5.3-4 different microsatellites can be multiplexed in each lane or
capillary by using 3-4 different fluorescent dyes.
6.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).
7.Sequence at least one copy of each allele to verify allele sizes.

22. Size Mother Baby “Father”
Hypothetical gel pattern
for microsatellite heterozygous
for all individuals.

23. Paternity Analyses & Conclusions :
1.Baby and mother are expected to share on allele, and the baby and
father the other allele.
2.If baby and father do not share a common allele, the “father” is not
the father.
3.If the baby and father do share a common allele, paternity is
possible, but not proven, because other men in the population also
carry the allele at some frequency.
4.Frequency of alleles that are shared in common by chance can be
calculated, and an appropriate number of microsatellites analyzed to
calculate probability of paternity.
5.To achieve high probability, 6-12 loci should be assayed (exact
number depends on variation in population for each marker).
6.If each locus has few alleles, more loci are required. If allelic
diversity if high, fewer loci can be analyzed.

24. Single nucleotide polymorphisms (SNPs) :
1.DNA sequences of most individuals are almost identical, >99%.
2.Single base pair differences occur about once every 500-1000 bp.
3.In most populations there is a common SNP, and several less
common SNPs.
4.About 3 million SNPs occur in the human genome, and these are
becoming popular genetic markers.
5.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.

25. How to type SNPs:
1.SNPs can be typed by hybridizing a complementary oligonucleotide
(e.g., single-base extension assay ).
2.If the stringency is high (i.e., temperature), the oligonucleotide will
fail to bind to DNAs showing polymorphism.
3.Many hundreds of SNPs can be tested simultaneously using:
DNA microarrays (DNA-chips, Gene-Chips, SNP-chips)
First developed in the early 1990s.
Ordered grid of short, complementary, known sequence
oligonucleotides placed at fixed positions on silicon, glass, or
nylon substrate.
Oligonucleotides are experimentally determined and are either
(1) microspotted or (2) synthesized on the chip.
User defined SNP chips are available commercially, and can
contain >400,000 different probes.

26. Fig. 9.2, Typing a SNP with an oligonucleotide.

27. How to type SNPs (cont.) :
1.SNP chip is designed with an array of user defined oligonucleotides
attached to the substrate (the SNP chip is the probe).
2.Oligonucleotides match each of the common and variant alleles in
the population (all alleles of interest).
3.Target DNAs are labeled with a fluorescent tag and hybridized (or
not) to the chip.
4.Fluorescence pattern is detected by a laser.
5.Because the oligonucleotides are known, the pattern indicates the
type of alleles the individual possesses.
6.Many different alleles at thousands of different loci can be screened
simultaneously in the same experiment.

28. Fig. 9.5, Schematic of a SNP chip assay.