molecular biology – pcr, sequencing, genomics molecular biology techniques ii. polymerase chain...
TRANSCRIPT
MOLECULAR BIOLOGY – PCR, sequencing, Genomics
MOLECULAR BIOLOGY TECHNIQUES II.
Polymerase Chain Reacton – PCRDNA sequencing
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
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
~94oC - Denaturation step
~60oC - Primer annealing step
37oC - 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 Mg2+ 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
5’ 3’
5’3’
5’ 3’
5’3’5’ 3’
5’3’
DNApol
DNApol
primerprimer
DENATURATION 94°C
DENATURATION
DENATURATION
MOLECULAR BIOLOGY – PCR
ANNEALING ~60oC
dsDNA FRAGMENT TO BE AMPLIFIED
EXTENSION - 37oC (Klenow)
DENATURATION
With each repeated THERMAL CYCLE (denaturation, annealing & extension) the amount of target dsDNA doubles
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)
94oC37oC60oC
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 (50-80oC)
Has an extremly heat stable (t1/2 >40 mins at 95oC) DNA polymerase
Taq polymerase ideally suited to PCR!
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 100oC)
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 000Taq 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)
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 94-96oC 2-3 mins. ensures all template DNA is single stranded (some DNApol require ‘hot-start’ for activation e.g. Pfu)
DENATURATION 94-96oC 0.5-2 mins. longer denaturation will ensure more single stranded DNA and better efficiency at cost of enzyme stability
ANNEALING ~60oC 0.5-2 mins. Higher temperature increase product specificity (less chance of mismatches forming) but lowers potential yield. 15-25oC < melting temperature Tm of annealed primer
EXTENSION ~72oC ~1 min/kb Taq processivity = 150 nucleotide per second (Pfu slower)
TERMINAL EXTENSION ~72oC 5-10 mins. Allows any incomplete products get finished
x25-30
Cetus Corporation
KARY B. MULLIS
1983 PCR discovery1985 published, patent pending1987 patented1993 Nobel prize
Journal of Molecular Biology Volume 56, Issue 2 , 14 March 1971, Pages 341-361
Studies on polynucleotidesXCVI. 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
‘Polymerase chain reaction (PCR)’ amplification of DNA -
video/ tutorial
http://www.sumanasinc.com/webcontent/animations/content/pcr.html
MOLECULAR BIOLOGY – PCR
MOLECULAR BIOLOGY – PCR
Experimental uses of PCRIntroduction 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
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 DQ
5‘ TGCTGTGATGT GCTGATGCTGAATGC 3‘T
Mutagenic primer
Experimental uses of PCR
MOLECULAR BIOLOGY – PCR
Degenerate PCR
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
GCTGTGATGTAGCTGATGCTGAAT3’TCGATCGCACGACACTACATCGACTACGACTTAAGACGCTACAA’5
GCTGTGATGTAGCTGATGCTGAATG3’TCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAA’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
Inverse PCR
MOLECULAR BIOLOGY – PCR
A method to amplify a particular DNA region (e.g. containing a gene) with only partial
sequence information
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)
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
CCGAGTAGCTAGGAACTGATGAATGTCGATCGCACGACACTACATCGACTACGACTTAAGACGCTACAATCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAATTGAGGTCGATGA...CCCCATGAGGGTGTGACCCGACATGACATGACATTGAGGCACAAATCAATGTAGA
AAAAAAAAAAAAAAAAAAAAAAAAA
MOLECULAR BIOLOGY – PCR
5’
Experimental uses of PCRReverse Transcription PCR (RTPCR)
3’
mRNA
cDNATTTTTTTTTTTTTTTTTTTTTTTTTCTACATTGATTTGTGCCTCAATGTCATGTCATGTCGGGTCACACCCTCATGGGG. . .
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 maybe required
Real-time PCR (Quantitative PCR or Q-PCR)
General PCR kinetics
PCR cycles
pro
du
ct
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’
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
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
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
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
http://www.scanelis.com/webpages.aspx?rID=679
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)
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?
dGTP
dTTP
dATP
dCTP
ddGTP
MOLECULAR BIOLOGY – sequencing
Dideoxynucleotide trisphosphate chain terminator/ Sanger DNA sequencing
Target DNA, oligonucleotide primer & DNApol
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’
5’-CTGGGATACTGTACTAGC-3’
DNApol
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’
5’-CTGGGATACTGTACTAGC
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’
5’-CTGGGATACTGTACTAGC
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’
5’-CTGGGATACTGTACTAGC
3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’
5’-CTGGGATACTGTACTAGC
ACTTAACCTTTG
ACTTAACCTTTGATCG
ACTTAACCTTTGATCGATCTAG
ACTTAACCTTTGATCGATCTAGCCG
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
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
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
-
+ AACACTACTTACTTAACTTAA
ACTTAACCTTTGATCGATCTAGCCG
ACTTAACCACTTAAC
ACTTAACCT
ACTTAACCTTTGATCACTTAACCTTTGATACTTAACCTTTGAACTTAACCTTTGACTTAACCTTTACTTAACCTT
ACTTAACCTTTGATCG
ACTTAACCTTTGATCGATCTA
ACTTAACCTTTGATCGATCTACTTAACCTTTGATCGATCACTTAACCTTTGATCGATACTTAACCTTTGATCGA
ACTTAACCTTTGATCGATCTAGCCACTTAACCTTTGATCGATCTAGCACTTAACCTTTGATCGATCTAG
‘Dideoxynucleotide trisphosphate chain
terminator/ Sanger DNA sequencing’ principle - videos/
tutorials
http://spine.rutgers.edu/cellbio/assets/flash/dideoxy.htm http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf
MOLECULAR BIOLOGY – sequencing
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 tubingdetector
The specific fluorescence signature of each band informs which nucleotide is at that position in the target DNA
MOLECULAR BIOLOGY – sequencing
How to sequence a human genome - video/ tutorial
http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/Animations/DNA/WTDV026689.htm
Featuring a description of automated fluorescence based DNA sequencing
Why not try to deduce the sequence of larger segments of DNA . . .
MOLECULAR BIOLOGY – PCR, sequencing
Genes . . .
Chromosomal regions . . .
Whole Chromosomes . . .
Entire genomes?
1990 Human Genome Project(HGP)
Complete sequencing of the whole human genome within 15 years
MOLECULAR BIOLOGY – PCR, sequencing
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)
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
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 richin 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
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
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
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)
How to sequence a human genome by shotgun sequencing - video/
tutorial
http://www.genome.gov/19519278#al-3
MOLECULAR BIOLOGY – Genome sequencing
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
MOLECULAR BIOLOGY – PCR, sequencing
Illumina based DNA sequencing
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 is therefore primed for copying resulting in a copy of the sample DNA being
immobilised to the flow cell surface (the original sample DNA is washed away)
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
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’
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
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!
The principles of ‘illumina-based’ next generation based sequencing -
video
MOLECULAR BIOLOGY – PCR, sequencing
http://www.illumina.com/technology/sequencing_technology.ilmn
MOLECULAR BIOLOGY – PCR, sequencing
http://www.youtube.com/watch?v=77r5p8IBwJk
The principles of ‘illumina-based’ next generation DNA sequencing -
video
ION PERSONAL GENOME MACHINE SEQUENCER
http://lifetech-it.hosted.jivesoftware.com/videos/1016
NextGen DNASeq Ion Torrent - video/ tutorial
Craig Venter Institute Sorcerer II expedition
MOLECULAR BIOLOGY – PCR, sequencing
„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