Aydemir Akin, Ching Ching Wu*, Tsang long Lin

1. Amplification and cloning of infectious bursal disease virus genomic RNA segments by long and accurate PCR Aydemir Akin, Ching Ching Wu*, Tsang long Lin Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette Received in revised form 17 May 1999; accepted 18 May 1999

2. Abstract • RNA denaturation by heating • RTase lacking RNase-H activity • RNase-H digestion • Long and accurate PCR(LA-PCR) • Utility for any IBDV strains or isolates

3. Table of contents • 1. Introdction • 2. Material and methods • 2.1. Virus • 2.2. Extraction of viral RNA • 2.3 Oligodeoxynuclotide primers • 2.4. Reverse transcription and LA-PCR • 2.5. Cloning of cDNA amplicons of small and large IBDV genome segments • 2.6 nucleotide sequencing • 3. Results and discussion • 3.1. Extraction of viral dsRNA and synthesis of cDNA • 3.2. LA-PCR amplification

4. Introduction • Birnaviridae • Aquabirnavirus (e.g. infectious pancreatic necrosis virus) • Entomobirnavirus (e.g. drosophila-X virus) • Avibirnavirus (e.g. IBDV) • Immunosuppression • Two serotype • Nonenveloped and icosohedral shaped virion • Two double-stranded RNA segment

5. Duodenum Jejunum caecum Liver bursa of Fabricius 64-72 hrs Clinical disease and death Pathogenesis

6. Old method • Whole genome separated into smaller overlapping fragments • Screened and sequenced

7. Resembled by RE digestion • Subcloning into plasmid

8. Method detail • PCR • TA-cloning approach by Taq • But the disadvantage is 2 kb limitation

9. Improvement • Up to 42 kb • Additions of proof reading activity (3'-5' exonuclease) enzyme into PCR mixture • RNA Digestion of RNA-DNA hybrid by RNase-H • Use RTase lacking RNase-H activity

10. Viral RNA preparation • 3-week-old chickens orally infected • Bursae homogenization in PBS • Freeze-thawed three times • Proteinase K, 1% SDS, DEPC • KOAc, absolute ethanol, 14000 X g • Lithium chloride(2 and 4M) • Concentration measurement by spectrophotometry (260 nm)

11. primers

12. RT • dsRNA denaturation by heating • Superscript II system (MMLV RTase, 45℃, 60 min) • RNase-H digestion • Inactivation

13. LA-PCR • Amount of cDNA • Removal of RNA:cDNA hybrids • Magnesium concentration • Duration of denaturation during PCR amplification • Different polymerase mixtures

14. Cloning • dATP + Taq for addition of A on 3' end • Screening with T7N and Sp6N primers, or segment-specific primers • Orientation and identity were confirmed with internal primer

15. Sequencing • Li-Cor long-read sequencing reaction kit

16. Result and discussion • Extraction of viral dsRNA and synthesis of cDNA • LA-PCR amplification

17. Result of preparation • Produced 1.98 mg of very good quality (260/280 1.8) • Only MMLV produced long cDNA in both specifically and non-specifically primed cDNA.

18. Discussion of preparation • Random hexamers better than virus specific primers • RTase lacking RNase-H activity enables the production of long cDNA transcripts. • RNase-H digestion; In PCR, RNA may be serve as template (It will reduce efficiency) • Denaturation by heating without adding dimethyl-sulphoxide(DEPC) or methymercury hydroxide

19. Result of LA-PCR • Taq : Pfu = 64:1 (v/v) • These sequences obtained from IBDV strain E have the identity of IBDV. 3182 2777

20. Q&A