Genomics and Applied Biology 2016, Vol.7, No.5, 1-8
2
The HBV Creg DNA fragment from nt1087 to nt2488 including DR region, X gene and C gene that will be cloned
into eukarya expression vector pcDNA3.1(+) is in the DNAmodel graph below that was illustrated as Figure 1.
Figure 1 HBV genome structure
Note: From nt1235 to nt 2452, it includes X gene, C gene, X promoter, Enhancer
Ⅰ
,Enhancer
Ⅱ
,C promoter and DR region. DR
region contained DR1 and DR2 which is related to replication of HBV and has relevant to integration of HBV into host genome
2 Materials and Methods
Primer design
:
The primers were designed on the basis of HBV ayr subtype genome from gene bank (I
D: 5536), using the software primer5, synthesized by Invitrogen company. EcoR I and Nde I restriction
sites were added on 5' end sequences of forward and reverse primers respectively, P1F 5'-AGTGTAT
CA
TATG
CTTTCACTTTCTCGCCAACTTAC-3'(1087-1111), P1R 5'-GCCTACAGCCTCCTAGTACAAAGACC
TT-3'(1760-1788), P2F 5'-GTTAAAGGTCTTTGTACTA
G
GAGGCTGTAGG-3'(1757---1788), P2R 5'-TG
GA
ATTCC
AGTAAAGTTTCCCACCTTATGAGT-3'(2462-2488), the outline and italic letters were restriction s
ites.
DNA extraction and PCR amplification
:
Hepatitis B virus DNA was extracted from HBsAg, HBeAg and anti -
HBc positive serum of HBV infected patient, and then submitted to recombinant PCR amplification. PCR reaction
was carried out for amplification of HBV nt1087-2488 DNA fragment.
The PCR reaction can be described as:
The first PCR reaction
:
The PCR reaction contained 10 μl of DNA, 0.05 mM dNTP, 5 μl 10×PCR Buffer (with
Mg2+), 10 pico moles from each of HBV specific forward and reverse primers (The nt1087-1788 PCR reaction
use primer p1F and p1R, the nt1788-2488 PCR reaction use primer p2F and p2R), 2.5 unit of Taq DNA
polymerase in 50 μl final volume.
PCR reaction was carried out within 30 cycles: denaturation at 94
℃
for 30 sec, annealing at 56
℃
for 30 sec, and
elongation at 72
℃
for 50 sec.
Electrophoresis and DNA purification
:
PCR product was submitted to electrophoresis using 1% agarose gel,
stained by ethidium bromide (EB) and visualized under ultraviolet light (UV Trans illuminator), and then
recovered by gel DNA extraction kit (TIANGEN Cat. No I7804).
The second PCR reaction
:
The PCR reaction contained 0.05 mM dNTP, 5μl 10×PCR Buffer (with Mg2+), 10
pico moles from each of HBV nt1087-1788 PCR purified product and nt1788-2488 PCR purified product, 2.5 unit
of Taq DNA polymerase in 50 μl final volume.
PCR reaction was carried out within 20 cycles: denaturation at 94
℃
for 30 sec, annealing at 56
℃
for 30 sec, and
elongation at 72
℃
for 50 sec.
