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MOLECULAR BIOLOGY – PCR, sequencing, Genomics

MOLECULAR BIOLOGY – PCR, sequencing, Genomics. MOLECULAR BIOLOGY TECHNIQUES II. Polymerase Chain Reacton – PCR DNA sequencing. MOLECULAR BIOLOGY – PCR. Synthetically derived DNA. Cloning and/ or isolation from a genomic library. Amplification of specific DNA fragments.

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MOLECULAR BIOLOGY – PCR, sequencing, Genomics

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

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

  3. MOLECULAR BIOLOGY – PCR • 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 Polymerase Chain Reaction (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 THERMAL CYCLING ~94oC - Denaturation step ~60oC - Primer annealing step 37oC - Extension step 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 * 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

  4. MOLECULAR BIOLOGY – PCR dsDNA FRAGMENT TO BE AMPLIFIED 5’ 5’ 5’ 3’ 3’ 3’ DENATURATION94°C 3’ 3’ 3’ 5’ 5’ 5’ DENATURATION EXTENSION - 37oC (Klenow) DNApol ANNEALING ~60oC primer primer DNApol DENATURATION DENATURATION With each repeated THERMAL CYCLE (denaturation, annealing & extension) the amount of target dsDNA doubles

  5. MOLECULAR BIOLOGY – PCR Primitive PCR machine (3 water baths) DNApol (Klenow fragment) is killed by the heat INITIAL DENATURATION DENATURATION ANNEALING EXTENSION TERMINAL EXTENSION THERMAL CYCLING INITIAL DENATURATION DENATURATION e.g. x30 94oC 60oC 37oC Expensive Klenow had to be added after every thermal cycle ! Yellowstone National Park Thermal Springs 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! PCR’s DNApol problem !

  6. MOLECULAR BIOLOGY – PCR DNA polymerase error rate (misincorporated nucleotide) Klenow 1: 50 000 Taq polymerase 1: 9 000 Pfu polymerase 1: 1 300 000 ! ! ! 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) 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 Crucially Pfu polymerase has proof-reading activity and has the lowest error rate of any known thermostable polymerase 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 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 A typical PCR protocol Template DNA, sequence specific sense and antisense oligonucleotide primers, thermo-stable DNApol (e.g. Taq or Pfu), dNTPs & PCR buffer

  8. MOLECULAR BIOLOGY – PCR ‘Invention’ of PCR Journal of Molecular BiologyVolume 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.  KARY B. MULLIS Cetus Corporation H.G. Khorana Dr. Kjell Kleppe 1983 PCR discovery 1985 published, patent pending 1987 patented 1993 Nobel prize 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

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

  10. MOLECULAR BIOLOGY – PCR PCR Incorporation of useful DNA sequence into PCR product EPITOPE TAG Addition of extra protein coding DNA sequence for a ‘tag’ that can be used experimentally to detect or purify a protein Generation of restriction enzyme sites for cloning 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

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

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

  13. MOLECULAR BIOLOGY – 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 Experimental uses of PCR

  14. MOLECULAR BIOLOGY – PCR Experimental uses of PCR Detecting SNPs by PCR G GCTGTGATGTAGCTGATGCTGAAT 3’TCGATCGCACGACACTACATCGACTACGACTTAAGACGCTACAA’5 SNP-specific primer amplification GCTGTGATGTAGCTGATGCTGAATG 3’TCGATCGCACGACACTACATCGACTACGACTTACGACGCTACAA’5 CTGCGATGTT SNP • 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. MOLECULAR BIOLOGY – PCR 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 Unknown DNA can know be PCR amplified using primers specific to the known sequence at each end DNA SEQUENCE WILL REVEAL WHERE UNKNOWN FRAGMENTS WHERE ORIGINALLY LIGATED (i.e. LEFT AND RIGHT) Inverse PCR DNA digested with restriction enzyme not cutting in known region 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 Generated compatible ends are ligated into a circle DNA re-linearised by digestion with a restriction enzyme recognising a site within the know sequence

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

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

  18. MOLECULAR BIOLOGY – PCR General PCR kinetics product Plateau due to exhaustion of reagents 2. 1. Measurements of abundance must be taken in the exponential phase of the PCR PCR cycles 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 Real-time PCR (Quantitative PCR or Q-PCR) Continuous measurement of product synthesis would be preferable i.e measurements in ‘real time’

  19. 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 Under PCR cycling conditions extension 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 Real-time PCR (Quantitative PCR or Q-PCR)

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

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

  22. 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

  23. MOLECULAR BIOLOGY – sequencing DNA 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)

  24. MOLECULAR BIOLOGY – 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 Dideoxynucleotide trisphosphate chain terminator/ Sanger DNA sequencing Sanger realised such ‘chain termination’ could be exploited to reveal the sequence of a specific/ target DNA molecule, but how?

  25. MOLECULAR BIOLOGY – sequencing DNApol 5’-CTGGGATACTGTACTAGC-3’ 3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’ ddGTP is radioactively labelled 5’-CTGGGATACTGTACTAGC 5’-CTGGGATACTGTACTAGC 5’-CTGGGATACTGTACTAGC 5’-CTGGGATACTGTACTAGC 3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’ 3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’ 3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’ 3’-GGACCCTATGACATGATCGATGAATTGGAAACTAGCTAGATCGGCACGAG-5’ Target DNA, oligonucleotide primer & DNApol Dideoxynucleotide trisphosphate chain terminator/ Sanger DNA sequencing ACTTAACCTTTG ACTTAACCTTTGATCG dGTP ACTTAACCTTTGATCGATCTAG ddGTP dTTP ACTTAACCTTTGATCGATCTAGCCG dATP dCTP 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)

  26. MOLECULAR BIOLOGY – sequencing G A T C dGTP dGTP dGTP dGTP ddCTP ddTTP ddGTP ddATP dTTP dTTP dTTP dTTP dATP dATP dATP dATP dCTP dCTP dCTP dCTP Target DNA, oligonucleotide primer & DNApol Target DNA, oligonucleotide primer & DNApol Target DNA, oligonucleotide primer & DNApol Target DNA, oligonucleotide primer & DNApol 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

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

  28. MOLECULAR BIOLOGY – sequencing ‘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

  29. MOLECULAR BIOLOGY – sequencing The specific fluorescence signature of each band informs which nucleotide is at that position in the target DNA Automatic DNA sequence analyzers Principle of automated DNA sequencing capillary electrophoretic tubing detector 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)

  30. MOLECULAR BIOLOGY – sequencing How to sequence a human genome - video/ tutorial Featuring a description of automated fluorescence based DNA sequencing http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/Animations/DNA/WTDV026689.htm

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

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

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

  34. MOLECULAR BIOLOGY – PCR, sequencing Whole Genome Shotgun DNA Sequencing Mapped BACs (i.e. in correct order on chromosome) Fragmentation of BAC clones and BAC sub-clone libraries (typically cloned into bacteriophage; ~2Kb) 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 Publication of a draft sequence in 2000 and a complete sequence in 2003 ! Human genome rich in repetitive sequences: ??? AAAAAAAAAAAAAAAAAAAAAAAA GTCCTGCATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAGCTTGGCTCACATAGT J. Craig Venter Francis Collins President William J. Clinton 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. 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. MOLECULAR BIOLOGY – PCR, sequencing J. Craig Venter – founder of ‘CELERA Genomics’ 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 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 the genome was ‘won’ for all of humanity and not for ‘profit’ !

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

  40. MOLECULAR BIOLOGY – PCR, sequencing NEXT GENERATION DNA SEQUENCING (NextGen DNASeq) Ultra high throughput with many millions of sequence reads per reaction allowing genomic scale experimentation analysis in single experiments! Examples of NextGen DNASeq technologies • 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

  41. MOLECULAR BIOLOGY – PCR, sequencing DNA or cDNA 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) Illumina based DNA sequencing Adapters ligated to ends of fragmented (~300bp) DNA sample DNA sample preparation 2-step process: 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) Specific DNA sequence adapters

  42. MOLECULAR BIOLOGY – PCR, sequencing Illumina based DNA sequencing 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, Bridge amplification 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 Repeated cycles of bridge amplification lead to the generation of copied complementary clusters of the original sample DNA 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. Cluster formation The flow cell surface is covered in several million dense clusters - all representing one original DNA molecule in the sample Actual sequence reaction utilizing ‘reversible chain terminator fluorescent dNTPs’

  44. MOLECULAR BIOLOGY – PCR, sequencing 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 Illumina based DNA sequencing 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 Sequencing DNA clusters one base at a time Depending on the first nucleotide in the cluster, a specific fluorescent reversible chain terminator dNTP is incorporated leading to a stop in DNA synthesis!

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

  46. MOLECULAR BIOLOGY – PCR, sequencing The principles of ‘illumina-based’ next generation based sequencing - video http://www.illumina.com/technology/sequencing_technology.ilmn

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

  48. ION PERSONAL GENOME MACHINE SEQUENCER

  49. NextGen DNASeq Ion Torrent - video/ tutorial http://lifetech-it.hosted.jivesoftware.com/videos/1016

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