BIOTEHNOLOĢIJA III : REKOMBINANTU BIOTEHNOLOĢIJA JAUNĀ BIOTEHNOLOĢIJA I. Muižnieks, 2015. g. pavasaris.

BIOTEHNOLOĢIJA III :

REKOMBINANTU BIOTEHNOLOĢIJA

JAUNĀ

BIOTEHNOLOĢIJA

I. Muižnieks, 2015. g. pavasaris

Jaunās klonēšanas un in vivo gēnu inženierijas metodes

Klonēšanas metodes:

Gibsona savākšanās

In vivo gēnu inženierijas metodes:

Zn-pirkstu nukleāzes

TALEN nukleāzes

CRISP/Cas sistēma

Sekvenēšanas metožu attīstība

W-H Chen, Z-J Qin, J Wang,G-P Zhao. The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly. Nucleic Acids Research, 2013, 1–9, doi:10.1093/nar/gkt122

H. Kim & J-S Kim. A guide to genome engineering with programmable nucleasesNature Reviews Genetics 15, 321–334, (2014) doi:10.1038/nrg3686

a | Nuclease-induced double-strand breaks (DSBs) can lead to sequence insertion, nucleotide correction or change (red box) through homology-directed repair (HDR) in the presence of a donor DNA or a single-strand oligodeoxynucleotide (ssODN), both of which contain homology arms. DSBs can also be repaired through error-prone non-homologous end-joining (NHEJ), which does not require donor DNA or ssODN and consequently often leads to small insertions and deletions (indels). Typical indel sequences and the number of inserted (+3 and +1) or deleted (−2, −4 and −10) bases are shown. b | When two DSBs are generated in cis on a single chromosome by programmable nucleases, the flanking region can be deleted or inverted. c | When two DSBs are generated on two different chromosomes, chromosomal translocations can be induced.

Jaunās pieejas augu genoma modifikācijai

Zn-pirkstu nukleāzes

Genes VII, Lewin B., 2005, p. 276/382

Jaunās pieejas augu genoma modifikācijai

Zn-pirkstu nukleāzes

Zinc-finger nucleases as gene therapy agents, D Carroll, 2008

Modificēta FokI endonukleāze

A schematic representation of a zinc-finger nuclease (ZFN) pair

a | Each ZFN is composed of a zinc-finger protein (ZFP) at the amino terminus and the FokI nuclease domain at the carboxyl terminus. In the zinc-finger motif consensus, X represents any amino acid. Target sequences of ZFN pairs are typically 18–36 bp in length, excluding spacers.

b | A computer model structure of a ZFN pair bound to DNA is shown. Each zinc-finger is shown in shades of pink in ribbon (left) and space-filling (right) representations. The grey region represents the linker between the DNA-binding and catalytic domains. The FokI catalytic domains are shown in blue and purple at the centre using space-filling representations.

Part b is modified, with permission, from Ref. 191 © (2011) Genetics Society of America.

Transcription activator like effectors (TALEs) (Boch et al. 2009; Moscou and Bogdanove 2009).TALEs are produced by plant pathogens in the genus Xanthomonas, which deliver the proteins to plant cells during infection

Proteīna-DNS mijiedarbībasspecifiskums TALE efektorā

Jaunās pieejas augu genoma modifikācijai

TALE faktori

Christian M. et al., Targeting DNA Double-Strand Breaks with TAL Effector Nucleases, Genetics 186: 757–761 (October 2010)

A schematic representation of a transcription activator-like effector nuclease (TALEN) pair.

a | Each TALEN is composed of transcription activator-like effectors (TALEs) at the amino terminus and the FokI nuclease domain at the carboxyl terminus. Each TALE repeat is comprised of 33–35 amino acids and recognizes a single base pair through the amino acids at positions 12 and 13, which is called the repeat variable diresidue (RVD; shown in red). Target sequences of TALEN pairs are typically 30–40 bp in length, excluding spacers.

b | In the TALE–DNA co-crystal structure, the RVDs in TALE interact with DNA in the major groove. The amino-terminal repeats (designated as 0 and −1 in the box) contact 5′ thymine.

Part b is modified, with permission, from Ref. 73 © (2012) American Association for the Advancement of Science.

CRISPR (clustered regularly interspaced short palindromic repeat)Cas (CRISPR-associated) genes,

CRISPR-based adaptive immune systems Terns and Terns, 2011

Jaunās pieejas augu genoma modifikācijai

CRISP-Cas

Mali P. et al. RNA-Guided Human Genome Engineering via Cas9. Science, V339, p. 824, 2013

Jaunās pieejas genoma modifikācijai

CRISP-Cas

Schematic representations of RNA-guided engineered nucleases (RGENs).

a | An RGEN is comprised of CRISPR (clustered regularly interspaced short palindromic repeat)-associated protein 9 (Cas9), a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), which form the dualRNA–Cas9.

b | Alternatively, an RGEN can contain Cas9 and a single-chain guide RNA (sgRNA). The guide sequence in the crRNA (part a) or sgRNA (part b) is complementary to a 20-bp target DNA sequence known as protospacer, which is next to the 5′-NGG-3′ (where N represents any nucleotide) sequence known as protospacer adjacent motif (PAM). Grey dots indicate weak bonding.

c | Target DNA cleaved by an RGEN yielding blunt ends is shown.

d | A three-dimensional model of Cas9 complexed with DNA is shown. Part d courtesy of D. W. Taylor (University of California, Berkeley, USA), J. A. Doudna (University of California, Berkeley, USA) and M. Jinek (University of Zurich, Switzerland).

J. J. Day (2014) New approaches to manipulating the epigenome. www.dialogues-cns.org

DNS sekvenēšanas metožu attīstība

Walter Gilbert, 1932

Andrejs Mirzabekovs, 1937 -2002

DNS sekvenēšana ar daļēji specifiskas ķīmiskās degradācijas palīdzību

http://nationaldiagnostics.com/article_info.php/articles_id/20

Frederick Sanger, 1918

www.nwfsc.noaa.gov/.../figures/moranfig4.htm

METODES

Manuāla

sekvenēšana

Analīzes metodes

Klasiskā pieeja: fragmentu klonēšana, subklonēšana, sekvenēšana

http://seqcore.brcf.med.umich.edu/doc/

JAC, BAC, PAC, PUC

Bioinformātikas idejas: nejauša klonēšana (shotgun), sekvenēšana

http://img4.wikia.nocookie.net

K. Venters, B.Klintons, F. Kolins, 2000, cilvēka genoma projekta finiša taisnē

http://www.vfa-bio.de/static/

Resekvenēšanas metodes “Resekvenēšana”, vai genoma sekvenēšana nto reizi, vai genoma daļas sekvenēšana organismam, kam viena genoma sekvence jau zināma (vai pat radniecīgam organismam) ir vieglāka un lētāka nekā de novo sekvenēšana. Vairākas firmas piedāvā liela apjoma, ātrdarbīgas paralēlās resekvenēšanas platformas.

454 Life Sciences(http://www.454.com/enabling-technology/the-system.asp)Solexa (Illumina)(http://www.illumina.com/pages.ilmn?ID=203)

Samuel Levy, et al. (Craig Venter)

The Diploid Genome Sequence of an Individual Human

PLoS BIOLOGY October 2007 | Volume 5 | Issue 2113 10 | e254

David A. Wheeler, et al. (James Watson)

The complete genome of an individual by massively parallel DNA sequencing

Nature 452, 872-876 (17 April 2008)

Jeffrey M. Kidd, et al.

Mapping and sequencing of structural variation from eight human genomes

Nature 453, 56-64 (1 May 2008)

PERSONISKIE GENOMA PROJEKTI

Analīzes metodes

Analīzes metodes Solex – Illumina tehnoloģija

Analīzes metodesSolex – Illumina tehnoloģija

Analīzes metodesSolex – Illumina tehnoloģija

Solex – Illumina

JONU PUSVADĪTĀJU SEKVENĒŠANA –ION TORRENT TECHNOLOGIES

The Ion Proton™ Sequencer is ideal for sequencing both exomes — regions in the DNA that code for protein — and human genomes. The Ion Proton™ I Chip, ideal for sequencing exomes, will be available mid-2012. The Ion Proton™ II Chip, ideal for sequencing whole human genomes, will be available about six months later. In addition, the Ion Proton™ OneTouch™ system automates template prep and a stand-alone Ion Proton™ Torrent Server performs the primary and secondary data analysis.

January 10, 2012

Life Technologies Benchtop Ion Proton Sequencer will sequence human genomes in one day for less than $1000 by yearend and Illumina will have a competing sub-$1000 per human genome sequencer by yearend

Nanopore DNA sequencing technique promises entire genome in minutes or your money back

https://www.sciencenews.org/sites/

http://www.futuretimeline.net/blog/images/1137.jpg

Rick Merritt DNA Chip Will Plug Into Handsets 6/9/2014

Sekvenēšanas perspektīvas