HIV Drug Resistance Training
Module 3:
Principles of PCR and
HIVDR Sequencing
1
Topics




Isolation and Amplification of DNA
Sequencing
Identifying Mixtures
Lab Procedures
2
Objectives
Describe the PCR process.
Describe the importance of maintaining the
genetic diversity originally present in the sample.
 Identify lab procedures that maintain the genetic
diversity of the sample.
 Identify lab procedures that can maximize the
accuracy of genotyping results by minimizing
factors that might limit accuracy.


3
isolation and amplification of dna
How is the genetic sequence isolated and reproduced in large
enough quantities for testing?
Why is it important to maintain the genetic diversity of the sample?
4
HIV RNA Isolation
Lysis buffer
Virus sample
(plasma, DBS, etc)
Viral RNA and proteins
Plasma proteins, lipids, etc.
RNAses
Affinity
purification
Purified viral RNA
5
Amplification of PR/RT Sequences:
RT-PCR Overview
5’
gag
pol
env
3’
AAAAAA
Reverse primer
Forward primer
6
RT Reaction: First Strand Complimentary
DNA (cDNA) Synthesis
Viral RNA
5’
3’
A G U C A U C G C U A C G G A C U A G G C U U C G G A U C G G
A G C C T A G C C
T
G
A
A
3’
A
C
C
T
5’
G
C
A
primer
G
T
G
C
dNTPs
T
Reverse transcriptase
(e.g. MLV)
7
RT Reaction: First Strand Complimentary DNA
(cDNA) Synthesis
5’
3’
A G U C A U C G C U A C G G A C U A G G C U U C G G A U C G G
T C A G T A G C C A T G C C T G A T C C G A A G C C T A G C C
3’
primer
5’
8
PCR Reaction: Second Strand DNA Synthesis
A
T
G
A
A
C
5’
primer
3’
G
C
A
C
T
G
T
dNTPs
G
C
A G T C A T C G C
T C A G T A G C C A T G C C T G A T C C G A A G C C T A G C C
5’
3’
Thermostable DNA
polymerase (e.g. Taq)
9
PCR Reaction: Second Strand DNA Synthesis
5’
3’
primer
A G T C A T C G C T A C G G A C T A G G C T T C G G A T C G G
T C A G T A G C C A T G C C T G A T C C G A A G C C T A G C C
5’
3’
Thermostable DNA
polymerase (e.g. Taq)
10
PCR Reaction: Amplification of Patient Virus
PR/RT Sequences
5’
3’
A G T C A T C G C T A C G G A C T A G G C T T C G G A T C G G
Heat denaturation (95°C)
T C A G T A G C C A T G C C T G A T C C G A A G C C T A G C C
3’
5’
11
PCR Reaction: Amplification of Patient Virus
PR/RT Sequences
5’
3’
A G T C A T C G C T A C G G A C T A G G C T T C G G A T C G G
A G C C T A G C C
3’
5’
5’
3’
A G T C A T C G C
T C A G T A G C C A T G C C T G A T C C G A A G C C T A G C C
3’
5’
12
PCR Reaction: Amplification of Patient Virus
PR/RT Sequences
5’
3’
A G T C A T C G C T A C G G A C T A G G C T T C G G A T C G G
T C A G T A G C C A T G C C T G A T C C G A A G C C T A G C C
3’
5’
5’
3’
A G T C A T C G C T A C G G A C T A G G C T T C G G A T C G G
T C A G T A G C C A T G C C T G A T C C G A A G C C T A G C C
3’
5’
13
Thermal Cycling: Exponential
Copy Number Amplification
Denaturation
(95°C)
Primer
extension
(72°C)
Primer
annealing
(50-65°C)
14
"Nested" PCR (In-house Assays Only)
5’
gag
pol
env
3’
AAAAAA
Reverse primer 1
Forward primer 1
1st round PCR product
Reverse primer 2
Forward primer 2
2nd round PCR product
Pro: increased sensitivity/specificity
Cons: increased chance of amplification bias;
additional manipulation required
15
Dependence of Sensitivity of Detection of Minor
Variants and Input Viral Load

Assume that 200 µl plasma used for RNA
extraction, 25% used for RT-PCR; RT successful
for 20% of RNA molecules; minority variant
present at 30% of total
Viral load
(copies/ml)
RNA copies in
RT rxn
Amplifiable
genomes
Probability that
variant missed*
100,000
5,000
1000
<0.01%
10,000
500
100
3.5%
1000
50
10
42.3%
*assuming 20% sensitivity of assay
16
Representative Sampling
N particles
sampled*
50% mixture
R+S
1000
300
100
30
10
Probability that R
variant % is
between 20-80%
100%
100%
100%
98.1%
80.7%
*e.g. number of amplifiable cDNA molecules in PCR reaction
17
Representative Sampling
N particles
sampled*
25% mixture
R+S
1000
300
100
30
10
Probability that R
variant %
between 20-80%
99.9%
95.6%
81.4%
62.2%
45.5%
*e.g. number of amplifiable cDNA molecules in PCR reaction
18
RT-PCR and "Quasi-species"
AAAAA
AAAAA
AAAAAAAAA
AAAAAAAAA
AAAAAAAAA
AAAAA
AAAAAAAAA
AAAAAAAAA
AAAAAAAAA
AAAAAAAAA
AAAAA
viral RNA
57%/14%/14%/14%
AAAAA
DNA copies (PR andRT)
from the virus RNA
DNA for sequencing
60%/0%/20%/20%
19
Discussion
How is the genetic sequence isolated and
reproduced in large enough quantities for testing?
 Why is it important to maintain the genetic
diversity of the sample?

20
sequencing
What is the purpose of sequencing reactions?
What data is produced in sequencing reactions?
21
Sequencing Reactions: Overview
PR 1
RT 1
RT 240+
Total region length
900- 1200 bases
Reaction 1
Reaction 2
Reaction 3
Reaction 4
Reaction 5
Reaction 6
Sequencing reactions (4 to 6) are performed to cover the entire
region. Approximately 600 bases are detected from each
reaction. Reaction components are DNA, primer, polymerase,
deoxynucleotides, dideoxynocleotides, and buffer.
22
Sequencing Reaction: DNA Denaturation
Double stranded
DNA (PCR products)
95° C
23
Sequencing Reaction: Primer Annealing
1
2
3
Each primer is in
a separate
reaction tube
4
5
6
24
Sequencing Reaction: Primer Extension
Utilizes Taq Polymerase to bulid
complementary DNA strand
25
Sequencing Reaction: Chain Termination
Fluorescently-labeled
dideoxynucleotide
triphosphate (ddNTP)
26
Sequencing: Electrophoresis
T
T
A
A
G
G
G
G
T
T
A
G
C
C
A
T
A
T
Automated basecalling is performed by the analyzer.
T
A
Y
183
C
A
T
M
184
G
G
A
D
185
T
T
A
Y
183
C
G
T
V
184
G
G
A
D
185
T
27
Sequencing Data Analysis
28
Dye-primer Sequencing (TruGene®)
Dye-labeled primers used instead of labeled dideoxy NTPs
 Primers labeled with one of 2 dyes
 A forward and reverse labeled primer used in a
reaction with one of the (unlabeled) di-deoxy
NTPs

29
ABI Sequencing Instruments
30
Alternative Sequencing Instruments
Beckman CEQ8000
TruGene
31
Discussion


What is the purpose of sequencing reactions?
What data is produced in sequencing reactions?
32
primer design
How is the sequence of PCR and sequencing primers determined?
What approaches can be used to design primers that work across
multiple subtypes of HIV?
33
PCR Primers: General Guidelines
5’
primer
3’
A G T C A T C G C
T C A G T A G C C A T G C C T G A T C C G A A G C C T A G C C
5’
3’





Flanking region of interest plus >~50 nt spacer
15-25 nt long
G-C base pair at 3' end if possible
Compatible melting temperatures
Avoid self-complimentary sequences at 3' end
(lead to primer dimers)
AGTGACTCGCTAGCGC
34
Sequencing Primers
PR 1
RT 1
RT 240+
Flanking region of interest plus >~20 nt spacer
Evenly distributed across target sequence, ~400500 nt apart
 15-20 nt long


35
Cross-subtype primer design
Both PCR and sequencing primers should bind to
regions that are conserved across subtypes
 Avoid mismatches near the 3' end in particular
 Where necessary, incorporate degenerate bases
(e.g. R or Y) or synthesize alternate primers and
mix together in defined ratio

SEQ1 ACGTATCGATCTCTGATTATACTGCATCGATATACGATACTATC
SEQ2 -----------A---------------T---------------SEQ3 ---------C-C-------------------------A-----SEQ4 --------G--C-------------------------------SEQ5 -----------C---------------T---------A-----SEQ6 --------GC---A-------------T---------------Forward primer
CTGATTATACTGCATYGATATACG
36
37
Pairs Activity: Primer Design
38
39
identifying mixtures
What is base calling?
What are some of the challenges of base calling?
40
Guidelines for Base-Calling

Automated base-calling is not 100% accurate
– Accuracy decreases in areas of poor quality, high
background, or artifacts such as "dye blobs"
– Discrimination between "true" and "false" mixtures

General requirements:
– Overlapping sequence derived from both strands
(sense and anti-sense primers)
– Short regions of single-stranded coverage may be
acceptable if high quality, and subject to supervisor's
review
– Take local signal to noise ratio and patterns into
account
41
Examples of Mixtures
Mixture at 184 – M/V
42
Examples of Mixtures
43
Examples of Mixtures
44
Discussion


What is base calling?
What are some of the challenges of base calling?
45
lab procedures
What lab practices will help contribute to accuracy of HIVDR
genotyping?
46
Discussion
Why is it importance to maintain the genetic
diversity originally present in the sample?
 In general, what lab procedures can maximize
the accuracy of genotyping results by minimizing
factors that might limit accuracy?

47
Reflection

What do we need to do to make sure our lab has
good procedures in place to ensure the accuracy
of the test?
48
Summary




Isolation and Amplification of DNA
Sequencing
Identifying Mixtures
Lab Procedures
49