optimization of a polymerase chain reaction system …

Prince Jonathan Pappoe-Ashong1, Charles A. Brown2, Julius A. A. Mingle1, Kwamena K. W. C. Sagoe1

1University of Ghana, School of Biomedical and Allied Health Sciences, Department of Medical Microbiology

2University of Ghana, School of Biomedical and Allied Health Sciences, Department of Medical Laboratory Sciences

OPTIMIZATION OF A POLYMERASE CHAIN REACTION SYSTEM FOR WHOLE GENOME AMPLIFICATION OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 2 (HIV-2)

Introduction

Aim

Objectives

Results Results – con’t Results – con’t

Conclusions and Recommendations

References

Research has shown there is a global decline of HIV-2 prevalence in

countries which previously had a very high prevalence.

Therefore the need to preserve the genomes of available HIV-2 strains in

order to use genomics to understand the possible beneficial properties of

HIV-2.

In this study, attempts were made to amplify the whole genome of HIV-2

using polymerase chain reaction (PCR) from viral RNA and proviral DNA

extracted from archived human plasma and whole blood, respectively.

To develop a PCR system that can be used to amplify the entire genome of

all known subtypes and recombinant forms of HIV-2.

1. Identify and select primers that can be used for whole genome amplification

of HIV-2.

2. Optimize reagents, thermocycling conditions and primers for regular and long

range PCR amplification of whole genome HIV-2

Methodology Proviral DNA (from archived human whole blood) and viral RNA (from

archived human plasma) were extracted using Zymo Research Viral DNA kit

(Irvin, CA, U.S.A.) and Zymo Research Viral nucleic acids kit, respectively.

Primers that targeted the conserved sites of the 3’ and 5’ long terminal repeat

were selected based on in silico analyses using Snapgene® tool version 2.1.0

(Table 3), Los Alamos HIV database sequence locator and AnalyzAlign tools

and NCBI BLAST and Ensemble BLASTN/BLAT results (Table 4). Eight

overlapping primers for PCR whole genome amplification of HIV-2 were also

selected and used for overlap PCR amplification of whole genome of HIV-2

(Table 3, Figures 10 & 11) . Long range and overlapping PCR of whole genome

of HIV-2 were carried out using long range Kit (New England Biolabs Inc.,

Ipswich, MA, USA) and cDNA synthesized using NEB ProtoScript II (New

England Biolabs Inc., Ipswich, MA, USA) for proviral DNA and viral RNA

templates (Figures 8-11). The HIV-2 cDNA synthesis was optimized using (i)

Random primers, (ii) d(T)23 VN, and (iii) mixture of random primers and

d(T)23 VN in different cDNA synthesis reactions (Figure 9). Amplified whole

genome of HIV-2 was gel purified and PCR confirmed using two sets of

primers, ENVF/ENVG EB2/EB5 (Primers targeting highly conserved sites of

HIV-2 env gene the) and EB2/EB5 and gel electrophoresis.

Figure 1: Binding sites of the primers on the HIV-2 NC_001722.1 reference sequence genome

Primer Combinations PCR Fragment/s (In Silico) bp

*P1 (^F) 10,359 & 855

*P8 (#R)

HIV2upA (^F) 9349

HIV2lowA (#R)

HIV2upA1 (^F) 9180

HIV2low (#R)

*P1 $ (^F) 5811

*P4 $ (#R)

*P6 $ (^F) 4787

*P8 $ (#R)

*POL4F $ (^F) 4603

*ENV G $ (#R)

*Pol F $ (^F) 4001

*ENV G $ (#R)

*GAG A $ (^F) 4603

*P4 $ (#R)

Table 3: Primer combinations and their respective in silico amplification fragment lengths

*Novel HIV-2 primer combinations, ^F = forward primer, #R = reverse primer, $ = Overlapping PCR primers

Figure 2: P1 and P8 primers and their relationship

to the target HIV-2 NC_001722.1 reference

sequence genome

Figure 3: HIV2upA and HIV2lowA primers and their

relationship to the target HIV-2 NC_001722.1

reference sequence genome

Figure 4: P1 and P4 primers and their



Figure 5: GagA and P4 primers and their



Figure 7: PolF and EnvG primers and their

relationship to the target HIV-2

NC_001722.1 reference sequence genome

Proviral DNA and viral RNA were successfully extracted from archived whole

blood and plasma, respectively. Six primers (Table 1) were selected out of the 68

primer sequences retrieved using in silico analyses for long range single PCR

amplification of HIV-2 whole genome (Table 4). Primers P1/P8 and

HIV2upA/HIV2lowA were successfully used in the whole genome amplification

of HIV-2 (Figures 8 & 9). Overlapping primers P1/P4, P6/P8, PolF/EnvG and

Pol4F/EnvG covering the entire genome of HIV-2 were also successfully used in

the whole genome amplification of HIV-2 (Figures 10 & 11). The amplified

whole genome fragment was confirmed to be HIV-2 by PCR using primers

EB2/EB5 and ENVF/ENVG (Figure 13). Figure 6: Pol4F and EnvG primers and

their relationship to the target HIV-2

NC_001722.1 reference sequence genome

PRIMER

NAME

(F/R)

PRIMER POSITION IN

HIV-2 WHOLE GENOME

SEQUENCE

PRIMER

SEQUENCE

SIMILARI-

TY TO HIV

BEN REF

SEQ239

NUMBER OF VARIANTS

IN MAJOR HIV-2

SUBTYPES

NUMBER OF MUTATION IN

VARIANTS OF HIV-2 SUBTYPES

HIV-2

TARGETED

GENE

POSITION

IN GENOME

using

HXB2(bp)

A B G AB

H2_0

1_A

B

A B G AB H2_01_

AB

HIV2upA (F) 5' LTR 884→905 100% 1 1 1 1 2 2 2 2 2 2,3

HIV2lowA(R) 3' LTR 708→729 100% 2 2 1 NR 2 0,1 0,1 0 NR 0

HIV2upA1 (F) 5' LTR 889→908 ,

9850 → 9829 100% 2 1 1 1 1 0,1 2 2 2 2

HIV2lowB (R) 3' LTR

564 → 542,

10025 →

10003

100% 4 2 NR NR 1 0,2,

1,6 1,0 NR NR 1

P1 (F) 5' LTR 1→43 93% 8 4 NR NR 3

6,7,

21,2

,3,2,

4,5

7,6,

7,6 NR NR 6,7,5

P8 (R) 3' LTR 855 → 838,

9940 → 9923 100% 6 3 1 NR 1

0,1,

2,3,

1,1

0,1,

1 1 NR 0

Table 4: Summary of primer analysis using Los Alamos HIV Database Sequence locator and AnalyzAlign

*NR = Not represented

5 µL DNA template

used for PCR

2 µL DNA

template

used for PCR

Figure 8: Ethidium bromide stained 1% agarose

electrophoregram of long range PCR product using

proviral DNA as template and varying MgCl2

concentrations and template volumes. Lanes 1 and

11= negative controls, lanes 2 & 3 = Mg2+

concentration is too low, there either is no product

formed (lane 2) or very little product formed (lane

3). Lanes 4, 6, 7, 8 represent optimal

concentrations of Mg2+ for this PCR set ups

indicated by the presence of the 10000 bp amplicon

product. Lanes 5 and 10 are indicative of

excessively stringent conditions with no 10000 bp

product formed. Lane M = 1kb gel pilot (Qiagen,

Germany).

Figure 9: Ethidium bromide-stained 1.0%

agarose gel electrophoregram of amplified

whole genome of HIV-2 from first strand

cDNA using primers P1/P8. Lanes 1and 2 =

PCR fragment from d (T) V23 synthesized

cDNA; Lanes 3 and 4 = PCR fragment from

mixture of random primers and d (T) V23

synthesized cDNA; Lane 5 and 6 = PCR

fragment from random primers synthesized

cDNA, Lane M = 2log DNA ladder (New

England Biolabs Inc., Ipswich, MA, USA).

Figure 10: Ethidium bromide-stained 1.0% agarose gel

electrophoregram of overlap PCR amplification whole genome

of HIV-2 using proviral DNA as template and HIV-2 whole

genome overlapping primers. Lanes 1 and 2 = PCR product from

primers P1 / P4; Lanes 3 and 4 = PCR product from primers Gag

A / P4; Lanes 5 and 6 = PCR product from primers P6 / P8; Lane

M = 1kb extended DNA ladder (New England Biolabs Inc.,

Ipswich, MA, USA)


electrophoregram of overlap PCR amplification whole genome of

HIV-2 using proviral DNA as template and HIV-2 whole genome

overlapping primers. Lanes 1 and 2 = PCR product from primers

PolF/EnvG; Lanes 3 = PCR product from primers Pol4F/EnvG;

Lane M = 1kb extended DNA ladder (New England Biolabs Inc.,

Ipswich, MA, USA).

1 2 3 M


electrophoregram of PCR amplification confirmation of HIV-2

genome using HIV-2 primers ENVF/ENVG.

Lane M = 2log DNA ladder (NEW ENGLAND BIOLABS INC.,

IPSWICH, MA, USA). Lanes 1 and 2 shows expected 300 bp

fragments. Lanes 1 and 2 shows expected 300 bp fragments.

Lanes 3=Negative control.

Acknowledgements Noguchi Postdoctoral Secretariat mainly funded this work

Staff of Department of Medical Microbiology, UG

Dr. Mrs. Evelyn Bonney (Virology Unit, NMIR)

Dr. Gorge Kyei (Washington University, U.S.A.)

Staff of clinical virology Lab, UG

Mr. Isaac Boamah

Ms. Aba Hayford

Ms. Makafui Sheshie

This study has shown novel primer combinations of already existing HIV-2

primers which can be used for PCR amplification of the entire genome of HIV-

2. The six primers for long-range and six primers for overlapping amplification

of HIV-2 whole genome identified in this study may be useful in HIV-2

genotyping using viral RNA and proviral DNA. This can also help in generating

HIV-2 whole or partial genome sequences that can be used for analysis of HIV-2

drug resistance, evolution and recombination analysis of HIV-2 for both LTNP

and fast progressors since primers cover the env and pol genes.

It is therefore recommended that;

1. Future studies should be focused on whole genome sequencing and whole

genome analysis of both HIV-1 and HIV-2.

2. The extra bands observed in this study should also be sequenced to determine

the other possible priming sites of the primers used.

Damond, F., Loussert-Ajaka, I., Apetrei, C., Descamps, D., Souquiere, S., Lepretre, A., Simon, F.

(1998). Highly sensitive method for amplification of human immunodeficiency virus type 2 DNA.

Journal of Clinical Microbiology.

Lorenz, T. C. (2012). Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and

Optimization Strategies. Journal of Visualized Experiments, (63), e3998.

http://doi.org/10.3791/3998

Novitsky, V., Zahralban-Steele, M., McLane, M. F., Moyo, S., van Widenfelt, E., Gaseitsiwe, S.,

Essex, M. (2015). Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis

of HIV Drug Resistance and HIV Clustering. Journal of Clinical Microbiology, 53(8), 2581–92.

http://doi.org/10.1128/JCM.00756-15.

Roux, K. H. (2009). Optimization and troubleshooting in PCR. Cold Spring Harbor Protocols, 4(4),

1–7. http://doi.org/10.1101/pdb.ip66.

10

kb

1 kb

1 2 3 4 5 6 7 8 9 10 11 M

10 kb

4kb

M 1 2 3 4 5 6

1 2 3 4 5 6 M

0.3

kb

0.5

kb

3kb

1 2 3 M

3kb

M 1 2 3

Step Temperature

/Time

Number

of Cycles

Initial Denaturation 94oC/ 5 min 1

Denaturation 94oC/ 75 sec

45 Annealing 52oC/ 75 sec

Extension 68oC/ 15 min

Final extension 68oC/ 20 min 1

Table 1: Long range PCR reaction using

synthesized first strand cDNA/Proviral DNA

as template thermocycling conditions

Component Tube

1

Tube

2

Tube

3

Tube

4

Tube

5

Tube

6

Tube

7

Tube

8

Tube

9

DNA

template

(µL)

5 5 5 5 5 5 2 2 2

Additional

MgCl2

(mM)

- 0.5 1.0 1.5 2.0 2.5 0.5 1.0 1.5

Table 2: Long range PCR reaction optimization

http://doi.org/10.3791/3998

http://doi.org/10.3791/3998

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http://doi.org/10.3791/3998

http://doi.org/10.3791/3998

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optimization of a polymerase chain reaction system …

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