Modified Terminal Deoxynucleotidyl Transferase (TdT) Enzymes

1 . A modified terminal deoxynucleotidyl transferase (TdT) enzyme comprising amino acid modifications when compared to a wild type sequence SEQ ID NO 1 or a truncated version thereof or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species or the homologous amino acid sequence of Polµ, Polβ, Polλ, and Polθ of any species or the homologous amino acid sequence of X family polymerases of any species, wherein the amino acid modifications are one or more of the amino acid changes E385N, P422S or R442Q.

2 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to claim 1 wherein the modification is E385N.

3 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to claim 1 or claim 2 wherein the modification is P422S.

4 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 1 to 3 wherein the modification is R442Q.

5 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 1 to 4 wherein having a further modification selected from M152T, T160R, E174S, C179A, C179T, C179E, C179G, M183L, M183Q, M183E, M183C, M183N, A195S, A195T, S198N, D210V, Q211R, Q224L, S245G, S245P, R259H, H263L, H263R, H263Q, H263K, L265P, L265V, A273G, H275Q, L285M, A293V, G303S, Q304L, L312Q, A314S, I318L, G328A, C331Y, C331R, V335A, V335C, M344V, V348H, R357M, D368K, D368R, D368H, C381S, F390Y, K392M, K401T, A404V, V424F, V424I, E441C, R445H, K453N, N458E, Y462F, K464T, D488Q, D488V or D488A.

6 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to claim 5 wherein the modification is D368H.

7 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 5 or 6 wherein the modification is G328A.

8 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 5 to 7 wherein the modification is M152T.

9 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 5 to 8 wherein the modification is Y462F.

10 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 5 to 9 wherein the modification is C381S.

11 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 5 to 10 wherein the modification is I318L.

12 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 5 to 11 wherein the modification is A195T.

13 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 5 to 12 wherein the modification is V424I.

14 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 5 to 13 wherein the modification is H275Q.

15 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 5 to 14 wherein the modification is C179A.

16 . The modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 1 to 15 wherein the enzyme is truncated.

17 . A modified terminal deoxynucleotidyl transferase (TdT) enzyme according to any one of claims 1 to 16 wherein the wild type sequence is selected from

gi|768

Bos taurus

gi|460163

Gallus gallus

gi|494987

Xenopus laevis

gi|1354475

Oncorhynchus mykiss

gi|2149634

Monodelphis domestica

gi|12802441

Mus musculus

gi|28852989

Ambystoma mexicanum

gi|38603668

Takifugu rubripes

gi|40037389

Raja eglanteria

gi|40218593

Ginglymostoma cirratum

gi|46369889

Danio rerio

gi|73998101

Canis lupus familiaris

gi|139001476

Lemur catta

gi|139001490

Microcebus murinus

gi|139001511

Otolemur garnettii

gi|148708614

Mus musculus

gi|149040157

Rattus norvegicus

gi|149704611

Equus caballus

gi|164451472

Bos taurus

gi|169642654

Xenopus (Silurana) tropicalis

gi|291394899

Oryctolagus cuniculus

gi|291404551

Oryctolagus cuniculus

gi|301763246

Ailuropoda melanoleuca

gi|311271684

Sus scrofa

gi|327280070

Anolis carolinensis

gi|334313404

Monodelphis domestica

gi|344274915

Loxodonta africana

gi|345330196

Ornithorhynchus anatinus

gi|348588114

Cavia porcellus

gi|351697151

Heterocephalus glaber

gi|355562663

Macaca mulatta

gi|395501816

Sarcophilus harrisii

gi|395508711

Sarcophilus harrisii

gi|395850042

Otolemur garnettii

gi|397467153

Pan paniscus

gi|403278452

Saimiri boliviensis boliviensis

gi|410903980

Takifugu rubripes

gi|410975770

Felis catus

gi|432092624

Myotis davidii

gi|432113117

Myotis davidii

gi|444708211

Tupaia chinensis

gi|460417122

Pleurodeles waltl

gi|466001476

Orcinus orca

gi|471358897

Trichechus manatus latirostris

gi|478507321

Ceratotherium simum simum

gi|478528402

Ceratotherium simum simum

gi|488530524

Dasypus novemcinctus

gi|499037612

Maylandia zebra

gi|504135178

Ochotona princeps

gi|505844004

Sorex araneus

gi|505845913

Sorex araneus

gi|507537868

Jaculus jaculus

gi|507572662

Jaculus jaculus

gi|507622751

Octodon degus

gi|507640406

Echinops telfairi

gi|507669049

Echinops telfairi

gi|507930719

Condylura cristata

gi|507940587

Condylura cristata

gi|511850623

Mustela putorius furo

gi|512856623

Xenopus (Silurana) tropicalis

gi|512952456

Heterocephalus glaber

gi|524918754

Mesocricetus auratus

gi|527251632

Melopsittacus undulatus

gi|528493137

Danio rerio

gi|528493139

Danio rerio

gi|529438486

Falco peregrinus

gi|530565557

Chrysemys picta bellii

gi|532017142

Microtus ochrogaster

gi|532099471

Ictidomys tridecemlineatus

gi|533166077

Chinchilla lanigera

gi|533189443

Chinchilla lanigera

gi|537205041

Cricetulus griseus

gi|537263119

Cricetulus griseus

gi|543247043

Geospiza fortis

gi|543351492

Pseudopodoces humilis

gi|543731985

Columba livia

gi|544420267

Macaca fascicularis

gi|545193630

Equus caballus

gi|548384565

Pundamilia nyererei

gi|551487466

Xiphophorus maculatus

gi|551523268

Xiphophorus maculatus

gi|554582962

Myotis brandtii

gi|554588252

Myotis brandtii

gi|556778822

Pantholops hodgsonii

gi|556990133

Latimeria chalumnae

gi|557297894

Alligator sinensis

gi|558116760

Pelodiscus sinensis

gi|558207237

Myotis lucifugus

gi|560895997

Camelus ferus

gi|560897502

Camelus ferus

gi|562857949

Tupaia chinensis

gi|562876575

Tupaia chinensis

gi|564229057

Alligator mississippiensis

gi|564236372

Alligator mississippiensis

gi|564384286

Rattus norvegicus

.

18 . A method of nucleic acid synthesis, which comprises the steps of:

(a) providing an initiator oligonucleotide;

(b) adding a 3′-blocked nucleotide to said initiator oligonucleotide in the presence of a terminal deoxynucleotidyl transferase (TdT) as defined in any one of claims 1 to 17 ;

(c) removal of all reagents from the initiator oligonucleotide;

(d) cleaving the blocking group in the presence of a cleaving agent; and

(e) removal of the cleaving agent.

19 . The method as defined in claim 18 , wherein greater than 1 nucleotide is added by repeating steps (b) to (e).

20 . The method as defined in claim 18 or claim 19 wherein the 3′-blocked nucleotide is blocked with a group selected from 3′-O-azidomethyl, 3′-aminooxy, 3′-O-(N-oxime), 3′-O-allyl 3′-O-cyanoethyl, 3′-O-acetyl, 3′-O-nitrate, 3′-phosphate, 3′-O-acetyl levulinic ester, 3′-O-tert butyl dimethyl silane, 3′-O-trimethyl(silyl)ethoxymethyl, 3′-O-ortho-nitrobenzyl, or 3′-O-para-nitrobenzyl.

21 . The method as defined in claim 20 wherein the 3′-blocked nucleoside is blocked by either a 3′-O-azidomethyl, 3′-aminooxy or 3′-O-allyl group.

22 . A kit comprising a terminal deoxynucleotidyl transferase (TdT) as defined in any one of claims 1 to 17 in combination with an initiator oligonucleotide and one or more 3′-blocked nucleoside triphosphates.