1. PCR AND DNA SEQUENCING
MBG-487
Işık G. Yuluğ

2. Polymerase Chain Reaction (PCR)
DNA melting
Primer annealing
Nobel Prize in Chemistry 1993,
at age 48
DNA elongation
Kary Mullis
(invented PCR in 1983)

3. Exponential nature of PCR amplification

4. PCR
Every cycle results in a doubling of the number of strands DNA present
After the first few cycles, most of the product DNA strands made are the same length as the distance between the primers
The result is a dramatic amplification of a the DNA that exists between the primers. The amount of amplification is 2 raised to the n power; n represents the number of cycles that are performed. After 20 cycles, this would give approximately 1 million fold amplification. After 40 cycles the amplification would be 1 x 1012

6. Try for equal Tm for both primers

7. Avoid primer dimer formation
Marginally problematic primer

8. Use Software to avoid of such problems

9. Typical PCR gel (Every PCR should by gel-verifyed)

10. PCR can be very tricky Optimizing PCR protocols
While PCR is a very powerful technique, often enough it is not possible to achieve optimum results without optimizing the protocol
Critical PCR parameters:
- Concentration of DNA template, nucleotides, divalent cations (especially Mg2+) and polymerase
- Error rate of the polymerase (Taq, Vent exo, Pfu)
- Primer design

11. Primer design General notes on primer design in PCR
Perhaps the most critical parameter for successful PCR is the design of primers
Primer selection
Critical variables are:
- primer length
- melting temperature (Tm)
- specificity
- complementary primer sequences
- G/C content
- 3’-end sequence
Primer length
- specificity and the temperature of annealing are at least partly dependent on primer length
- oligonucleotides between 20 and 30 (50) bases are highly sequence specific
- primer length is proportional to annealing efficiency: in general, the longer the primer, the more inefficient the annealing
- the primers should not be too short as specificity decreases

12. Primer design Specificity
Primer specificity is at least partly dependent on primer length: there are many more unique 24 base oligos than there are 15 base pair oligos
Probability that a sequence of length n will occur randomly in a sequence of length m is:
Example: the mtDNA genome has about 20,000 bases, the probability of randomly finding sequences of length n is:
n Pn
x 10-2
x 10-5
P = (m – n +1) x (¼)n

13. Primer design Complementary primer sequences
primers need to be designed with absolutely no intra-primer homology beyond 3 base pairs. If a primer has such a region of self-homology, “snap back” can occur
- another related danger is inter-primer homology: partial homology in the middle regions of two primers can interfere with hybridization. If the homology should occur at the 3' end of either primer, primer dimer formation will occur
G/C content
ideally a primer should have a near random mix of nucleotides, a 50% GC content
there should be no PolyG or PolyC stretches that can promote non-specific annealing
3’-end sequence
- the 3' terminal position in PCR primers is essential for the control of mis-priming
- inclusion of a G or C residue at the 3' end of primers helps to ensure correct binding (stronger hydrogen bonding of G/C residues)

14. Primer design
Melting temperature (Tm)
- the goal should be to design a primer with an annealing temperature of at least 50°C
- the relationship between annealing temperature and melting temperature is one of the “Black Boxes” of PCR
- a general rule-of-thumb is to use an annealing temperature that is 5°C lower than the melting temperature
- the melting temperatures of oligos are most accurately calculated using nearest neighbor thermodynamic calculations with the formula:
Tm = H [S+ R ln (c/4)] – °C log 10 [K+]
(H is the enthalpy, S is the entropy for helix formation, R is the molar gas constant and c is the concentration of primer)
- a good working approximation of this value can be calculated using the Wallace formula:
Tm = 4x (#C+#G) + 2x (#A+#T) °C
- both of the primers should be designed such that they have similar melting temperatures. If primers are mismatched in terms of Tm, amplification will be less efficient or may not work: the primer with the higher Tm will mis-prime at lower temperatures; the primer with the lower Tm may not work at higher temperatures.

15. Fidelity of PCR is often an issue

16. Proof-reading activity enzymes

18. If complete copies is amplified

21. LIST OF PCR REACTION CONDITIONS THAT MUST BE OPTIMIZED
PRIMER ANNEALING TEMPERATURE:
Increase in temperature: Increases specificity of primer annealing by destabilizing base pair mismatches.
Decrease in temperature: Increases the sensitivity (and yield) of the reaction by stabilizing base pairing.

22. LIST OF PCR REACTION CONDITIONS THAT MUST BE OPTIMIZED
DNA POLYMERASE:
Enzyme concentration: Enzyme concentrations affect the sensitivity and specificity; too little enzyme produces insufficient product and too much enzyme decreases specificity.
Type of DNA polymerase: Taq enzyme is the most efficient enzyme but it has also the highest error rate; in contrast, pfu has a decreased error rate but synthesizes the least amount of product.

23. LIST OF PCR REACTION CONDITIONS THAT MUST BE OPTIMIZED
MAGNESIUM CONCENTRATION:
Varying the (MgCl2): Low MgCl2 increases specificity, high MgCl2 stabilizes primer annealing and increases sensitivity, but can also decrease primer specificity.

24. LIST OF PCR REACTION CONDITIONS THAT MUST BE OPTIMIZED
CYCLE PARAMETERS:
Denaturation temperature: Elevated denaturation temperature can increase sensitivity by allowing complete template denaturation, especially of G+C rich targets; however, Taq polymerase activity decreases rapidly above 93oC.
Duration time of primer extension: Longer primer extension times increase sensitivity in long distance PCR.
Cycle number: Assay sensitivity is determined by both the efficiency of the enzyme reaction and the initial number of DNA target molecules; it should be necessary to increase sycle number beyond 35 if the reaction contains <103 initial target molecules.

27. Non-specific PCR and how to improve it+ G L Y M A R K E
Increase in Mg concentraton 5% D M S O
Just
PCR

28. -- generates PCR products with single A overhangs on the 3´-ends
PCR enzymes
Taq DNA polymerase, the first enzyme used for PCR,
is still the most popular.
-- high processivity and is the least expensive choice
Halflife at 95C is 1.6 hours
-- generates PCR products
with single A overhangs on the 3´-ends
(Suitable for TOPO-cloning)
“Topo” cloning system (Invitrogen)

29. The technology behind TOPO Cloning
The key to TOPO Cloning is the enzyme, DNA topoisomerase I,
which functions both as a restriction enzyme and as a ligase.
Its biological role is to cleave and rejoin DNA during replication.
Vaccinia virus topoisomerase I specifically recognizes the pentameric
sequence 5’-(C/T)CCTT-3’ and forms a covalent bond with the
phosphate group of the 3’ thymidine.
It cleaves one DNA strand, enabling the DNA to unwind.
The enzyme then religates the ends of the cleaved strand and
releases itself from the DNA.
To harness the religating activity of topoisomerase, TOPO vectors are
provided linearized with topoisomerase I covalently bound to
each 3’ phosphate.
This enables the vectors to readily ligate DNA sequences with
compatible Ends.
In only 5 minutes at room temperature, the ligation is complete
and ready for transformation into E. coli.

30. From Invitrogen

31. Tth polymerase Mg 2+ Mn 2+ Thermus thermophilus strain HB8.
RNA-dependent DNA-polymerase activity in the presence of Mn2+ ions.
Mg 2+
Mn 2+
DNA-dependent DNA-polymerase activity in the presence of Mg2+ ions.
The fragment should be ideally smaller 1 kb.

32. Pfu polymerase more expensive (from Pyrococcus furiosus).
Proofreading or high fidelity DNA polymerases
(from Pyrococcus furiosus).
approx.1 / 2, 000,000 nucleotides before making an error.
In comparison Taq DNA polymerase
makes an error in approx. every 1/ 10,000 nucleotides.
can tolerate temperatures exceeding 95°C,
enabling it to PCR amplify GC-rich targets.
more expensive

33. Vent (From Thermococcus litoralis)
also known as Tli polymerase
Very termostable: Halflife at 95oC is approximately 7 hours
3'->5' exonuclease activity presents
Vent error rate is intermediate between Taq and Pfu.
2-5 x 10-5 errors/bp
Other polymerases:
Deep Vent (Pyrococcus species GB-D) (New England Biolabs)
New England Biolabs claims fidelity is equal to or greater than that of Vent.
Replinase (Thermus flavis) 1.03 x 10-4 errors/base

34. Long-Range PCR Use of two polymerases:
a non-proofreading polymerase Taq
is the main polymerase in the reaction,
a proofreading polymerase (3' to 5' exo) Pwo
is present at a lower concentration.
22-24 kb PCR products achieved on
Qiagen and Eppendorf PCR mixes
Taq+ Pwo
(Pyrococcus woesei) ;
Pwo is very stable,
2 hrs at 100 C

35. DNA SEQUENCING

36. DNA sequencing: Importance
Basic blueprint for life
Gene and protein
Function
Structure
Evolution
Genome-based diseases- “inborn errors of metabolism”
Genetic disorders
Genetic predispositions to infection
Diagnostics
Therapies

37. DNA sequencing methodologies: 1977!
Maxam-Gilbert
base modification by general and specific chemicals.
depurination or depyrimidination.
single-strand excision.
not amenable to automation
Sanger
DNA replication.
substitution of substrate with chain-terminator chemical.
more efficient
Automation *

38. Maxam-Gilbert ‘chemical’ method

39. “bio” based methods
Sanger
dideoxynucleotides

40. DNA chemistry

41. DNA biochemistry: replication fork

42. SEQUENCING: (Sanger method)
Frederick Sanger
(Nobel prize 1980 with Paul Berg and Walter Gilbert)

43. DNA replication: biochemistry5’
purine or
pyrimidine P O OH P O OH P O OH HO C N O
purine or
pyrimidine O N O P O C O OH 3’ OH

44. Dideoxynucleotide blocks chain elongation

45. DNA sequencing: Sanger-II
purine or
pyrimidine P O OH P O OH P O OH HO C N O
purine or
pyrimidine O
chain
termination
method N O P O C O OH H

46. Sanger method

47. Methods of sequence visualization:
1. Labeled primer
2. Labelled DNA chain
(randomly)
3. Labeled terminators

48. Labelled nucleotide (radioactively)

49. Fluorescent DNA labeling with BigDye

50. Applied Biosystems Inc
Applied Biosystems Inc., have designed an automated method that combines the PCR and actual sequencing
<

51. DNA sequencing: chemistry* * * * * * * * * * * * * *

52. DNA sequencing: in practice
template + polymerase + 1
dCTP
dTTP
dGTP
dATP
ddATP
primer 2
dCTP
dTTP
dGTP
dATP
ddGTP
primer 3
dCTP
dTTP
dGTP
dATP
ddTTP
primer 4
dCTP
dTTP
dGTP
dATP
ddCTP
primer
electrophoresis
A•T
G•C
T•A
C•G
extension

53. DNA sequencing: upgrade, second iteration, terminator-label
Disadvantages of primer-labels:
four reactions
tedious
limited to certain regions, custom oligos or
limited to cloned inserts behind ‘universal’ priming sites.
Advantages:
Solution:
fluorescent dye terminators

54. DNA sequencing: chemistry
template + polymerase +
dCTP
dTTP
dGTP
dATP
ddATP
ddGTP
ddTTP
ddCTP
electrophoresis
A•T
G•C
T•A
C•G
extension

55. DNA sequencing: photochemistry

56. DNA sequencing: Computation

57. DNA sequencing: Computation

58. Nucleotides for Sequencing
Standard nucleotides (A,T,C, G)
Modified versions of these nucleotides
Labeled so they fluoresce
Structurally different so that they stop DNA synthesis when they are added to a strand

59. Reaction Mixture Copies of DNA to be sequenced Primer DNA polymerase
Standard nucleotides
Modified nucleotides

60. Reactions Proceed
Nucleotides are assembled to create complementary strands
When a modified nucleotide is included, synthesis stops
Result is millions of tagged copies of varying length

61. Recording the Sequence
T C C A T G G A C C
T C C A T G G A C
Recording the Sequence
T C C A T G G A
T C C A T G G
T C C A T G
T C C A T
T C C A
electrophoresis
gel
T C C
DNA is placed on gel
Fragments move off gel in
size order; pass through
laser beam
Color each fragment
fluoresces is recorded on
printout
T C
one of the many fragments of
DNA migrating
through the gel T
one of the DNA fragments
passing through a laser beam
after moving through the gel
T C C A T G G A C C A

62. DNA Sequencing Goal: Find the complete sequence of A, C, G, T’s in DNA
Challenge:
There is no machine that takes long DNA as an input, and gives the complete sequence as output
Can only sequence ~500 letters at a time

63. DNA sequencing – vectors
Shake
DNA fragments
Known
location
(restriction
site)
Vector
Circular genome
(bacterium, plasmid) + =

64. Different types of vectors
Size of insert
Plasmid
2,000-10,000
Can control the size
Cosmid
40,000
BAC (Bacterial Artificial Chromosome)
70, ,000
YAC (Yeast Artificial Chromosome)
> 300,000
Not used much recently

65. DNA sequencing – gel electrophoresis
Start at primer (restriction site)
Grow DNA chain
Include dideoxynucleoside (modified a, c, g, t)
Stops reaction at all possible points
Separate products with length, using gel electrophoresis

66. Electrophoresis diagrams

67. Challenging to read answer

68. Challenging to read answer

69. Challenging to read answer

70. Reading an electropherogram
Filtering
Smoothening
Correction for length compressions
A method for calling the letters – PHRED
PHRED – PHil’s Read EDitor (by Phil Green)
Based on dynamic programming
Several better methods exist, but labs are reluctant to change

71. Output of PHRAP: a read A read: 500-700 nucleotides
A C G A A T C A G …A
…21
Quality scores: -10log10Prob(Error)
Reads can be obtained from leftmost, rightmost ends of the insert
Double-barreled sequencing:
Both leftmost & rightmost ends are sequenced

72. Method to sequence longer regions
genomic segment
cut many times at random (Shotgun)
Get one or two reads from each segment
~500 bp
~500 bp

73. Reconstructing the Sequence (Fragment Assembly)
reads
Cover region with ~7-fold redundancy (7X)
Overlap reads and extend to reconstruct the original genomic region

74. Definition of Coverage
Length of genomic segment: L
Number of reads: n
Length of each read: l
Definition: Coverage C = n l / L
How much coverage is enough?
Lander-Waterman model:
Assuming uniform distribution of reads, C=10 results in 1 gapped region /1,000,000 nucleotides

75. Challenges with Fragment Assembly
Sequencing errors
~1-2% of bases are wrong
Repeats
false overlap due to repeat

76. Repeats Bacterial genomes: 5% Mammals: 50% Repeat types:
Low-Complexity DNA (e.g. ATATATATACATA…)
Microsatellite repeats (a1…ak)N where k ~ 3-6
(e.g. CAGCAGTAGCAGCACCAG)
Transposons
SINE (Short Interspersed Nuclear Elements)
e.g., ALU: ~300-long, 106 copies
LINE (Long Interspersed Nuclear Elements)
~500-5,000-long, 200,000 copies
LTR retroposons (Long Terminal Repeats (~700 bp) at each end)
cousins of HIV
Gene Families genes duplicate & then diverge (paralogs)
Recent duplications ~100,000-long, very similar copies

77. Strategies for whole-genome sequencing
Hierarchical – Clone-by-clone yeast, worm, human
Break genome into many long fragments
Map each long fragment onto the genome
Sequence each fragment with shotgun
Online version of (1) – Walking rice genome
Start sequencing each fragment with shotgun
Construct map as you go
Whole Genome Shotgun fly, human, mouse, rat, fugu
One large shotgun pass on the whole genome

78. Hierarchical Sequencing

79. Hierarchical Sequencing Strategy
a BAC clone
map
genome
Obtain a large collection of BAC clones
Map them onto the genome (Physical Mapping)
Select a minimum tiling path
Sequence each clone in the path with shotgun
Assemble
Put everything together

80. Methods of physical mapping
Goal:
Map the clones relative to one another
Use the map to select a minimal tiling set of clones to sequence
Methods:
Hybridization
Digestion

81. 1. Hybridization p1 pn
Short words, the probes, attach to complementary words
Construct many probes p1, p2, …, pn
Treat each clone Ci with all probes
Record all attachments (Ci, pj)
Same words attaching to clones X, Y  overlap

82. 2. Digestion
Restriction enzymes cut DNA where specific words appear
Cut each clone separately with an enzyme
Run fragments on a gel and measure length
Clones Ca, Cb have fragments of length { li, lj, lk }  overlap
Double digestion:
Cut with enzyme A, enzyme B, then enzymes A + B

83. Online Clone-by-clone The Walking Method

84. The Walking Method
Build a very redundant library of BACs with sequenced clone-ends (cheap to build)
Sequence some “seed” clones
“Walk” from seeds using clone-ends to pick library clones that extend left & right

85. Walking: An Example

86. Advantages & Disadvantages of Hierarchical Sequencing
ADV. Easy assembly
DIS. Build library & physical map;
redundant sequencing
Whole Genome Shotgun (WGS)
ADV. No mapping, no redundant sequencing
DIS. Difficult to assemble and resolve repeats
The Walking method – motivation
Sequence the genome clone-by-clone without a physical map
The only costs involved are:
Library of end-sequenced clones (cheap)
Sequencing

87. Walking off a Single Seed
Low redundant sequencing
Many sequential steps

88. Walking off a single clone is impractical
Cycle time to process one clone: 1-2 months
Grow clone
Prepare & Shear DNA
Prepare shotgun library & perform shotgun
Assemble in a computer
Close remaining gaps
A mammalian genome would need 15,000 walking steps !

89. Walking off several seeds in parallel
Efficient
Inefficient
Few sequential steps
Additional redundant sequencing
In general, can sequence a genome in ~5 walking steps,
with <20% redundant sequencing

90. Using Two Libraries
Most inefficiency comes from closing a small ocean with a much larger clone
Solution: Use a second library of small clones

91. Whole-Genome Shotgun Sequencing

92. Whole Genome Shotgun Sequencing
cut many times at random
plasmids (2 – 10 Kbp)
forward-reverse paired reads
known dist
cosmids (40 Kbp)
~500 bp
~500 bp