9040992.ppt PCR test molecular biology techniques

Pcr rna sequence

1. MOLECULAR BIOLOGY – PCR, sequencing, Genomics
MOLECULAR BIOLOGY TECHNIQUES II.
Polymerase Chain Reacton – PCR
DNA sequencing

2. Amplification of specific DNA fragments
MOLECULAR BIOLOGY – PCR
Cloning and/ or isolation from a genomic library
Synthetically derived DNA
Both possible but not the most convenient of methods e.g. cost and/ or labour
intensive

3. Polymerase Chain Reaction (PCR)
MOLECULAR BIOLOGY – PCR
A mechanism to exponentially amplify a specific DNA fragment in a test tube,
using the principles of specific DNA base-pairing and DNA replication and
employing these in repeated cycles
* The oligonucleotide primer sequences must be complementary to DNA sequence flanking the fragment to be amplified and
match with DNA sequence from the opposing strands of that fragment - see next slide
THERMAL CYCLING

~94
o
C - Denaturation step
~60
o
C - Primer annealing step
37
o
C - Extension step
• DNA containing fragment to be amplified
(e.g. genomic DNA or cDNA)
• Two oligonucleotide primers (ss) specific
to DNA sequence of desired fragment*
• Purified DNA polymerase (Klenow frag.)

• deoxyribonucleotide triphosphates
(dNTPs)
• Buffer solution (with required Mg
2+
and K
+

cations)
x25-35
REPEATED THERMAL CYCLING - initiates new rounds of DNA replication that
can use the products of the previous round as template, thus exponentially
amplifying the target DNA fragment

4. 5’ 3’
5’3’
5’ 3’
5’3’
5’ 3’
5’3’
DNApol
DNApol
primerprimer
DENATURATION 94°C
DENATURATION
DENATURATION
MOLECULAR BIOLOGY – PCR
ANNEALING ~60
o
C
dsDNA FRAGMENT TO BE AMPLIFIED
EXTENSION - 37
o
C (Klenow)
DENATURATION
With each repeated THERMAL CYCLE (denaturation, annealing & extension) the amount of target dsDNA doubles

5. Yellowstone National Park Thermal Springs
MOLECULAR BIOLOGY – PCR
PCR’s DNApol problem !
INITIAL DENATURATION
DENATURATION
ANNEALING
EXTENSION
TERMINAL EXTENSION
THERMAL CYCLING
e.g. x30
Primitive PCR machine (3 water baths)
94
o
C
37
o
C
60
o
C
INITIAL DENATURATION
DENATURATION
DNApol (Klenow fragment) is killed by the heat
Expensive Klenow had to be added after every thermal cycle !
Isolation of thermophillic bacteria:
Thermophillus aquaticus (50-80
o
C)
Has an extremly heat stable (t
1/2 >40 mins at
95
o
C) DNA polymerase
Taq polymerase ideally suited to PCR!

6. MOLECULAR BIOLOGY – PCR
Thermostable DNA polymerases and PCR
The isolation of Taq polymerase permitted the automation of PCR
thermal cycling as fresh DNApol did not need to be added after every
cycle !
HOWEVER: Taq polymerase lacks a proofreading activity (3‘-5‘
exonuclease) and high error rate
Stratgene inc. isolated a DNA polmerase from the hyperthermophilic
archae (primitive bacteria) Pyrococcus furiosus found in the marine
sediment associated with ocean thermal vents
Pfu polymerase is extremely heat stable (Pyrococcus furiosus optimum
growth temperature is 100
o
C)
Crucially Pfu polymerase has proof-reading activity and has the lowest
error rate of any known thermostable polymerase
DNA polymerase error rate (misincorporated nucleotide)
Klenow 1: 50 000
Taq polymerase 1: 9 000

Pfu polymerase 1: 1 300 000 ! ! !
Pfu polymerase is IDEALLY suited for PCR applications where high
fidelity amplification of DNA is required (although more expensive than
Taq polymerase)

7. MOLECULAR BIOLOGY – PCR
A typical PCR protocol
Template DNA, sequence specific
sense and antisense
oligonucleotide primers, thermo-
stable DNApol (e.g. Taq or Pfu),
dNTPs & PCR buffer
STEP TEMP TIME NOTES
INITIAL DENATURATION 94-96
o
C 2-3 mins. ensures all template DNA is single stranded (some DNApol
require ‘hot-start’ for activation e.g. Pfu)
DENATURATION 94-96
o
C 0.5-2 mins. longer denaturation will ensure more single stranded DNA
and better efficiency at cost of enzyme stability
ANNEALING ~60
o
C 0.5-2 mins. Higher temperature increase product specificity (less
chance of mismatches forming) but lowers potential yield.
5
o
C less than melting temperature T
m
of annealed primers
EXTENSION ~72
o
C ~1 min/kb Taq processivity = 150 nucleotide per second (Pfu slower)
TERMINAL EXTENSION ~72
o
C 5-10 mins. Allows any incomplete products get finished
x25-30

8. Cetus Corporation
KARY B. MULLIS
1983 PCR discovery
1985 published,
patent pending
1987 patented
1993 Nobel prize
Journal of Molecular Biology
Volume 56, Issue 2 , 14 March 1971, Pages 341-361
Studies on polynucleotides
XCVI. Repair replication of short synthetic DNA's
as catalyzed by DNA polymerases
K. Kleppe
‡
, E. Ohtsuka
§
, R. Kleppe
‡
, I. Molineux
||
and H. G. Khorana
||

Institute for Enzyme Research of the University of Wisconsin, Madison, Wisc. 53706, U.S.A.
Received 20 July 1970.
 
Dr. Kjell Kleppe H.G. Khorana
Mullins would have been ‘aware’ of the work of Kleppe and
Khorana. Although their method did not amplify DNA it is
generally accepted their research was a ‘primer’ for PCRs
discovery
MOLECULAR BIOLOGY – PCR
‘Invention’ of PCR

9. ‘Polymerase chain reaction (PCR)’
amplification of DNA - video/ tutorial
http://www.sumanasinc.com/webcontent/animations/content/pcr.html
MOLECULAR BIOLOGY – PCR

10. MOLECULAR BIOLOGY – PCR
Experimental uses of PCR
Introduction of specific and useful DNA sequences
Sequence specific (i.e. complementary) DNA oligonucleotide
primer with non-complementary yet useful 3’ sequence
Incorporation of useful DNA sequence into PCR product
PCR
Generation of restriction
enzyme sites for cloning
EPITOPE TAG
Addition of extra protein coding DNA sequence for
a ‘tag’ that can be used experimentally to detect or
purify a protein

11. Experimental uses of PCR
MOLECULAR BIOLOGY – PCR
Introduction of specific mutations within recombinant DNA ‘directed mutagenesis’
3‘ CGCACGACACTACATCGACTACGACTTACGACGCTACAAGTTCATGAC 5‘
Protein coding DNA sequence (cDNA)
R T T L H R L R L T T L Q V H D
Q
5‘ TGCTGTGATGT GCTGATGCTGAATGC 3‘
T
Mutagenic primer

12. Experimental uses of PCR
MOLECULAR BIOLOGY – PCR
Degenerate PCR

13. MOLECULAR BIOLOGY – PCR
Experimental uses of PCR
Nested PCR: two rounds of consecutive PCR using a second pair of primers with annealing sites
within the products produced by the first pair of primers
Some DNA fragments can sometimes be difficult to amplify by PCR - (potential secondary
structures or spurious products arising from primers binding other on-target DNA). Nested PCR
will increase the yield of true target DNA

14. GCTGTGATGTAGCTGATGCTGA AT
3’TCGATCGCACGACACTACATCGACTACGACT TAAGACGCTACAA’5
GCTGTGATGTAGCTGATGCTGA ATG
3’TCGATCGCACGACACTACATCGACTACGACT TACGACGCTACAA’5
SNP
MOLECULAR BIOLOGY – PCR
G
amplification
CTGCGATGTT
SNP-specific primer
Experimental uses of PCR
Detecting SNPs by PCR
Detection of SNPs is important for:
• diagnosing certain genetic diseases arising from ‘point mutation’ e.g. sickle cell anaemia
(Hb gene E6V)
• identifying linkage traits e.g. SNPs in the Apolipoprotein E are associated with increased risk
of Alzheimer’s diseas

15. Inverse PCR
MOLECULAR BIOLOGY – PCR
A method to amplify a particular DNA region
(e.g. containing a gene) with only partial
sequence information
(useful for e.g. detection of transposable
elements insertion sites)
N.B. relies on being able to cut DNA with
‘restriction’ enzymes that only cut at specific
DNA sequences - see lecture 8
DNA digested with restriction enzyme not
cutting in known region
Generated compatible ends are ligated into a circle
DNA re-linearised by
digestion with a
restriction enzyme
recognising a site
within the know
sequence
Unknown DNA can know be PCR amplified using
primers specific to the known sequence at each
end
Unknown DNA
can know be
PCR amplified
using primers
specific to the
known sequence
PREVIOUSLY UNKNOWN
DNA SEQUENCE CAN BE
DETERMINED BY
SEQUENCING FROM
KNOWN FLANKS
DNA SEQUENCE WILL REVEAL WHERE UNKNOWN FRAGMENTS WHERE ORIGINALLY LIGATED ( i.e. LEFT AND RIGHT)

16. MICROSATELLITE SEQUENCES
Sequence repeats:
(A)n
(CA)n
(CAG)n
(CAGT)n
5’ 3’
3’ 5’
5’ 3’
3’ 5’
a
b
Variable Number of Tandem Repeats (VNTR)
AFLP – amplified fragment length polymorphism
DNA fingerprinting
MOLECULAR BIOLOGY – PCR

17. CCGAGTAGCTAGGAACTGATGAATGTCGATCGCACGACACTACATCGACTACGACT TAAGACGCTACAATCGATCGCACGACACTACATCGA
CTACGACTTACGACGCTACAATTGAGGTCGATGA...CCCCATGAGGGTGTGACCCGACATGACATGACATTGAGGCACAAATCAATGTAGA
AAAAAAAAAAAAAAAAAAAAAAAAA
MOLECULAR BIOLOGY – PCR
5’
Experimental uses of PCR
Reverse Transcription PCR (RTPCR)
3’
mRNA
cDNA
TTTTTTTTTTTTTTTTTTTTTTTTTCTACATTGATTTGTGCCTCAATGTCATGTCATGTCGGGTCACACCCTCATGGGG. . .
TCATCGACCTCAATTGTAGCGTCGTAAGTCGTAGTCGATGTAGTGTCGTGCGATCGATTGTAGCGTCTTAAGTCGTAGTCGATGTAGTGTCG
TGCGATCGACATTCATCAGTTCCTAGCTACTCGG
TTTTTTTTTTT
Reverse transcription
5’
3’
Normal PCR
Presence of DNA product reveals presence of mRNA in the original sample
However, more quantitative rather than qualitative results may be required

18. Real-time PCR (Quantitative PCR or Q-PCR)
General PCR kinetics
PCR cycles
p
r
o
d
u
c
t
Plateau due to exhaustion of
reagents
MOLECULAR BIOLOGY – PCR
Measurements of abundance
must be taken in the exponential
phase of the PCR
1. 2.
If the number of PCR cycles used were not in the
exponential phase, one could mistake samples 1.
and 2. of being of equal concentration
Continuous measurement of product synthesis would be preferable i.e
measurements in ‘real time’

19. Real-time PCR (Quantitative PCR or Q-PCR)
MOLECULAR BIOLOGY – PCR
SYBR green-based Q-PCR assay
• ds DNA intercalating dye
• fluoresces green under blue light
• only emits fluorescence when bound
to double stranded DNA
denaturation
annealing
extension
Under PCR cycling conditions
SYBR green fluorescence can be measured at
the end of either the annealing* or extension
steps after every PCR cycle and used to
calculate the amount of DNA in the sample
* Measurements usually taken at the end of the primer annealing step

20. MOLECULAR BIOLOGY – PCR
‘Real-time PCR (Q-PCR)’ using SYBR
green-based assay - video/ tutorial
http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/real-time-pcr.html
click on this link
https://www.youtube.com/watch?v=3H9oabhqDAc

21. Real-time PCR (Quantitative PCR or Q-PCR)
MOLECULAR BIOLOGY – PCR
Fluorescent hybridisation probe based methods (e.g. TaqMan probes)
DNA sequence complementary to DNA
sequence of target molecule
Fluorescent
reporter group

22. Real-time PCR (Quantitative PCR or Q-PCR)
MOLECULAR BIOLOGY – PCR
Fluorescent hybridisation probe based methods (e.g. TaqMan probes)
DNA sequence complementary to DNA
sequence of target molecule
Fluorescent
reporter group Fluorescence quencher
+ other PCR reagents
At each ANNEALING step, probe and primers
hybridises with target/ product DNA
Molecular proximity of quencher prevents
reporter fluorescence
During EXTENSION step the annealed probe is
digested by Taq DNApol (5’ - 3’ exonuclease activity)
Reporter fluorescence no longer quenched and
used to quantify the DNA present

23. MOLECULAR BIOLOGY – PCR
‘Real-time PCR (Q-PCR)’ using
fluorescent molecular probes - video/
tutorial
http://www.biosearchtech.com/support/videos/real
-time-pcr-probe-animation-video.aspx

SYBR Green vs TaqMan probes rap battle:
https://www.youtube.com/watch?v=nKrJnc1xWb0

24. DNA SEQUENCING
MOLECULAR BIOLOGY – sequencing
(i.e. determining the order of the four possible deoxynucleotides in one of
the DNA strands and by inference the order on the other strand)

25. MOLECULAR BIOLOGY – sequencing
Dideoxynucleotide trisphosphate chain terminator/ Sanger DNA sequencing
DNA backbone comprises phosphodiester bonds between the
5’ and 3’ carbon atoms of the deoxyribose moeities of
consecutive deoxynucleotides
Addition of an additional deoxynucleotide to a growing DNA
strand, during DNA synthesis, requires a free 3’-OH group
However, incorporation of a chemically modified
dideoxynucleotide (ddNTP), lacking a 3’-OH group,
would prevent additional polymerisation and hence
TERMINATE DNA synthesis
Sanger realised such ‘chain termination’ could be exploited to reveal the sequence of a
specific/ target DNA molecule, but how?

26. dGTP
dTTP
dATP
dCTP
ddGTP
MOLECULAR BIOLOGY – sequencing
Dideoxynucleotide trisphosphate chain terminator/ Sanger DNA sequencing
Target DNA, oligonucleotide
primer & DNApol
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCAC -5’
5’-CTGGGATACTGTACTAGC-3’
DNApol
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCAC -5’
5’-CTGGGATACTGTACTAGC
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCAC -5’
5’-CTGGGATACTGTACTAGC
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCAC -5’
5’-CTGGGATACTGTACTAGC
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCAC -5’
5’-CTGGGATACTGTACTAGC
TACTTAACCTTTG
Generation of a series of differently sized fragments
synthesised from the target DNA molecule that all end with
radio-labelled dideoxy-G (specified by C in the target DNA)
ddGTP is radioactively
labelled
TACTTAACCTTT GATCG
TACTTAACCTTT GATCGATCTAG
TACTTAACCTTT GATCGATCTAGCCG

27. MOLECULAR BIOLOGY – sequencing
dGTP
dTTP
dATP
dCTP
ddGTP
Target DNA,
oligonucleotide primer &
DNApol
dGTP
dTTP
dATP
dCTP
ddATP
Target DNA,
oligonucleotide primer &
DNApol
dGTP
dTTP
dATP
dCTP
ddTTP
Target DNA,
oligonucleotide primer &
DNApol
dGTP
dTTP
dATP
dCTP
ddCTP
Target DNA,
oligonucleotide primer &
DNApol
G A T C
Repeat reaction using the three other radio-labelled ddNTPS
Now have a complete population of varying length DNA fragments (at one base-pair resolution),
derived from target DNA, that end with one of four radio-labelled dideoxynucleotides

28. MOLECULAR BIOLOGY – sequencing
G A T C
polyacrylamide DNA sequencing gelautoradiography film
Read off DNA sequence from bottom
to top (5’-3’ on newly synthesised
strand). Reverse complement for the
other strand
-
+
A
AC
ACT
ACTT
ACTTA
ACTTAA
ACTTAACCTTTGATCGATCTAGCC G
ACTTAACC
ACTTAAC
ACTTAACCT
ACTTAACCTTTGATC
ACTTAACCTTTGAT
ACTTAACCTTTGA
ACTTAACCTTTG
ACTTAACCTTT
ACTTAACCTT
ACTTAACCTTTGATCG
ACTTAACCTTTGATCGATCT A
ACTTAACCTTTGATCGATCT
ACTTAACCTTTGATCGATC
ACTTAACCTTTGATCGAT
ACTTAACCTTTGATCGA
ACTTAACCTTTGATCGATCTAGC C
ACTTAACCTTTGATCGATCTAG C
ACTTAACCTTTGATCGATCTA G

29. ‘Dideoxynucleotide trisphosphate
chain terminator/ Sanger DNA
sequencing’ principle - videos/
tutorials
http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDel
Genoma/animaciones/secuencia.swf

MOLECULAR BIOLOGY – sequencing
https://www.youtube.com/watch?v=vK-
HlMaitnE

30. MOLECULAR BIOLOGY – sequencing
Automation of the Sanger DNA sequencing method using fluorescently
labelled ddNTPs
Each ddNTP varient is conjugated to a specific fluorescent group
(ddGTP, ddCTP, ddATP and ddTTP) allowing the 4 reactions to be
pooled in one tube and the electrophoresed in the same lane
Process can be highly automated using ‘capillary tube
electrophoresis’ coupled to automatic fluorescence detectors
(~1Kb max)
Principle of automated DNA sequencing
Automatic DNA sequence analyzers
capillary electrophoretic tubing
detector
The specific fluorescence signature of each band informs which
nucleotide is at that position in the target DNA

31. Why not try to deduce the sequence of larger
segments of DNA . . .
MOLECULAR BIOLOGY – PCR, sequencing
Genes
Chromosomal regions
Whole Chromosomes
Entire genomes

32. 1990 Human Genome Project
(HGP)
Complete sequencing of the whole human genome within 15 years
MOLECULAR BIOLOGY – PCR, sequencing

33. MOLECULAR BIOLOGY – PCR, sequencing
Whole Genome Shotgun DNA Sequencing
Human genome (blood donors)
Mapping BACs to known sequence
markers (i.e. identify from what part
of the genome does the BAC come
from)?
Isolation of genomic DNA
Cloning of the genomic DNA fragments
(i.e. to build a genomic DNA library;
consisting of BACs - 200Kb)

34. MOLECULAR BIOLOGY – PCR, sequencing
Whole Genome Shotgun DNA Sequencing
Fragmentation of BAC clones
and BAC sub-clone libraries
(typically cloned into
bacteriophage; ~2Kb)
Mapped BACs (i.e. in correct order on
chromosome)
Sanger-based sequencing of
the sub-clones (from either
end)
Sequence alignment of overlapping sequences
from various subclones to reconstitute the
entire BAC DNA sequence

35. MOLECULAR BIOLOGY – PCR, sequencing
Whole Genome Shotgun DNA Sequencing
Repeated iterations of sub-clone sequencing (to give sequence depth i.e.
confidence) and BAC reconstitution, for all the BACS covering the entire
genome.
GTCCTGCATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAGCTTGGCTCACATAGT
???
Human genome rich
in repetitive sequences:
!
Publication of a draft sequence in 2000 and a complete sequence in 2003
Francis Collins J. Craig Venter
President William J. Clinton
Now many hundreds of different
species’ genomes have been
shotgun sequenced

36. MOLECULAR BIOLOGY – PCR, sequencing
The politics of sequencing the human genome !!!
Founded as an international publicly funded consortium effort to sequence all
the bases of the human genome with 15 years at a cost of $3 billion
Aimed to provide free and open
access to all the data as a
resource for research biologists
During the 1990’s a number of groups had placed patents on genes that they
had cloned, setting a commercial precedent/ incentive to whole genome
sequencing

37. J. Craig Venter – founder of ‘CELERA Genomics’
$$$$$
MOLECULAR BIOLOGY – PCR, sequencing
1998 launched a commercial bid to sequence human genome and secure gene patents
Thus, the start of a race to publish the complete genome sequence between Celera and the
publicly funded HGP begun. It was eventually decided that patents on genes were not legal
but both projects ended up publishing at the same time

38. MOLECULAR BIOLOGY – PCR, sequencing
How the genome was ‘won’ for all
of humanity and not for ‘profit’ !
Storage of the human genome DNA sequence (3.3
billion base-pairs)
3300 books of 1000 pages with 1000 bp
per page
1 data CD (786 Mb; 2bits per bp)

39. How to sequence a human genome by
shotgun sequencing - video/ tutorial
http://www.genome.gov/19519278#al-3
MOLECULAR BIOLOGY – Genome sequencing

40. NEXT GENERATION DNA SEQUENCING (NextGen DNASeq)
MOLECULAR BIOLOGY – PCR, sequencing
Ultra high throughput with many millions of sequence reads per reaction allowing genomic scale
experimentation analysis in single experiments!
• Illumina (Solexa) sequencing
• Ion semiconductor sequencing (e.g. Ion Torrent)
• Lynx Therapeutics' Massively Parallel Signature Sequencing (MPSS)
• Polony sequencing
• 454 pyrosequencing
• SOLiD sequencing
• Ion semiconductor sequencing (e.g. Ion Torrent)
• DNA nanoball sequencing
• Helioscope(TM) single molecule sequencing
• Single Molecule SMRT(TM) sequencing
• Single Molecule real time (RNAP) sequencing
• Nanopore DNA sequencing
• VisiGen Biotechnologies approach
Examples of NextGen DNASeq technologies

41. MOLECULAR BIOLOGY – PCR, sequencing
Illumina based DNA sequencing – sequencing by synthesis
DNA or
cDNA
Specific DNA sequence adapters
Adapters ligated to ends of fragmented (~300bp) DNA
sample
2-step process:
1.ligation of the same oligonucleotides to
both ends
2. PCR based amplification, adding unique DNA
sequence at each end (i.e. pink and blue in
figure)
DNA sample
preparation
Sample DNA
attachment to flow
cell surface
Sample DNA adapters base-pair with
complementary oligos fixed to the surface of the
flow cell (pink or blue)
The sample DNA copied from an attached primer
resulting in a copy of the sample DNA that
immobilised to the flow cell surface (the original
sample DNA is washed away)

42. MOLECULAR BIOLOGY – PCR, sequencing
Illumina based DNA sequencing
Bridge amplification
The adapter sequences (pink or blue) at the free
end of the immobilised copies of the sample
DNA are free to base-pair with other
neighbouring oligos that are fixed to the
surface of the flow cell
Such ‘bridge’ interactions prime another round of
DNA copying,
The result is two complementary copies of the
original sample DNA being immobilised to the
slide in proximity to each other

43. MOLECULAR BIOLOGY – PCR, sequencing
Illumina based DNA sequencing
Cluster formation
Repeated cycles of bridge amplification lead to
the generation of copied complementary
clusters of the original sample DNA
The flow cell surface is covered in several million
dense clusters - all representing one
original DNA molecule in the sample
The cluster contains copies of both strands of the
original DNA (i.e. it’s complementary).
Therefore prior to cluster sequencing one
strand is removed by cleaving with a
restriction enzyme that recognises a
sequence within either the pink or blue
adapter.
Actual sequence reaction utilizing ‘reversible chain terminator
fluorescent dNTPs’

44. MOLECULAR BIOLOGY – PCR, sequencing
Illumina based DNA sequencing
Sequencing DNA
clusters one base
at a time
A mix of sequencing primers (complementary to
one of the adapter sequences), DNA
polymerase and differentially fluorescent
labelled reversible chain terminator dNTPs
(A, C, T and G) are added to flow cell
Depending on the first nucleotide in the cluster, a
specific fluorescent reversible chain
terminator dNTP is incorporated leading to
a stop in DNA synthesis!
After washing unincorporated nucleotides away, a
laser excites the flow cell and detects which
of the four fluorescent chain terminator
dNTPs were incorporated in each cluster on
the flow cell. i.e. decodes the first
sequenced base
Once an image recording what was the first nucleotide to be
incorporated in each cluster has been taken, both the
fluorescent dyes and the blocking group that prevents
extension of the DNA are removed (hence ‘reversible chain
terminator dNTPs) and the cycle is repeated

45. MOLECULAR BIOLOGY – PCR, sequencing
Illumina based DNA sequencing
Sequential
sequencing rounds
one base at a time
Possible to get up to 50 base-pairs of good
sequence but there are millions of different
clusters!

46. The principles of ‘illumina-based’ next
generation based sequencing - video
MOLECULAR BIOLOGY – PCR, sequencing
http://www.illumina.com/technology/next-generation-sequencing.html
(video on the right side of the website)

47. MOLECULAR BIOLOGY – PCR, sequencing
http://www.youtube.com/watch?v=77r5p8IBwJk
The principles of ‘illumina-based’ next
generation DNA sequencing - video

48. ION PERSONAL GENOME MACHINE SEQUENCER

50. Ion torrent sequencing
At each time, a chip is flooded with a single nucleotide. If the nucleotide matches
the sequence, H+ is released and pH is changed. If it does not match the
sequence, pH is not changed. Change in the pH is measured.

51. https://www.youtube.com/watch?v=ZL7DXFPz8rU
https://www.youtube.com/watch?v=WYBzbxIfuKs
NextGen DNASeq Ion Torrent - video/
tutorial

52. Comparison of next-generation sequencing methods
Method Read length
Accuracy
(single read not
consensus)
Reads per runTime per run
Cost per 1
million bases (in
US$)
Advantages Disadvantages
Single-molecule
real-time
sequencing
(Pacific
Biosciences)
10,000 bp to
15,000 bp avg
(14,000 bp N50);
maximum read
length >40,000
bases
[61][62][63]
87% single-read
accuracy
[64]
50,000 per SMRT
cell, or 500–1000
megabases
[65][66]
30 minutes to 4
hours
[67]
$0.13–$0.60
Longest read
length. Fast.
Detects 4mC,
5mC, 6mA.
[68]
Moderate
throughput.
Equipment can
be very
expensive.
Ion
semiconductor
(Ion Torrent
sequencing)
up to 400 bp 98% up to 80 million2 hours $1
Less expensive
equipment. Fast.
Homopolymer
errors.
Pyrosequencing
(454)
700 bp 99.9% 1 million 24 hours $10
Long read size.
Fast.
Runs are
expensive.
Homopolymer
errors.
Sequencing by
synthesis
(Illumina)
50 to 300 bp 99.9% (Phred30)
up to 6 billion
(TruSeq paired-
end)
1 to 11 days,
depending upon
sequencer and
specified read
length
[69]
$0.05 to $0.15
Potential for high
sequence yield,
depending upon
sequencer model
and desired
application.
Equipment can
be very
expensive.
Requires high
concentrations of
DNA.
Sequencing by
ligation (SOLiD
sequencing)
50+35 or 50+50
bp
99.9% 1.2 to 1.4 billion1 to 2 weeks $0.13
Low cost per
base.
Slower than other
methods. Has
issues
sequencing
palindromic
sequences.
[70]
Chain
termination
(Sanger
sequencing)
400 to 900 bp99.9% N/A
20 minutes to 3
hours
$2400
Long individual
reads. Useful for
many
applications.
More expensive
and impractical
for larger
sequencing
projects. This
method also
requires the time
consuming step
of plasmid
cloning or PCR.

53. Craig Venter Institute
Sorcerer II expedition
MOLECULAR BIOLOGY – PCR, sequencing

54. „Our researchers discovered at least 1,800 new species and more than
1.2 million new genes from the Sargasso Sea“
Intensive horizontal gene transfer
MOLECULAR BIOLOGY – PCR, sequencing