Abstract
Summary: Amplification of DNA by polymerase chain reaction (PCR) is influenced by the homology of oligonucleotide primers with the DNA template. We have developed a procedure, termed anchored PCR, whereby nucleotide sequence alterations in the template can be directly related to the quantity of amplified product. Genetic variation in the human immunodeficiency virus HIV-1 has been studied using anchored PCR. In four field isolates of the virus, the 3′LTR was compared both by PCR analysis of DNA from virus cultures and DNA sequencing. DNA templates that matched the primers varied less than threefold in PCR product yield, whereas significant 3′end primer-template mispairing decreased PCR product 10- to 100-fold. Using these guidelines for genetic variability manifested through PCR, 40 PCR primers encompassing the GAG, ENV, and 3′LTR segments of the genome were used to compare sequential HIV-1 isolates from six patients. Some primers were apparently located in genomic regions without significant interisolate variability, as they yielded equivalent amounts of amplified DNA from all the isolates. The quantity of amplified DNA obtained with other primers varied 10- to 100-fold among patients, but was consistent for sequential isolates from an individual patient. Two African HIV-1isolates were readily distinguished from a panel of North American isolates by the same method. Systematic classification of HIV-1 genetic variants may be possible by anchored PCR.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1241-1250 |
| Number of pages | 10 |
| Journal | Journal of Acquired Immune Deficiency Syndromes |
| Volume | 4 |
| Issue number | 12 |
| State | Published - 1991 |
| Externally published | Yes |
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Keywords
- Genetic variation
- HIV-1
- PCR typing
ASJC Scopus subject areas
- Infectious Diseases
- Pharmacology (medical)
- Virology
- Immunology and Allergy
Cite this
Genetic comparison of human immunodeficiency virus (Hiv-1) isolates by polymerase chain reaction. / McCutchan, Francine E.; Sanders-Buell, Eric; Oster, Charles W.; Redfield, Robert R.; Hira, Subash K.; Perine, Peter L.; Ungar, Beth L P; Burke, Donald S.
In: Journal of Acquired Immune Deficiency Syndromes, Vol. 4, No. 12, 1991, p. 1241-1250.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Genetic comparison of human immunodeficiency virus (Hiv-1) isolates by polymerase chain reaction
AU - McCutchan, Francine E.
AU - Sanders-Buell, Eric
AU - Oster, Charles W.
AU - Redfield, Robert R.
AU - Hira, Subash K.
AU - Perine, Peter L.
AU - Ungar, Beth L P
AU - Burke, Donald S.
PY - 1991
Y1 - 1991
N2 - Summary: Amplification of DNA by polymerase chain reaction (PCR) is influenced by the homology of oligonucleotide primers with the DNA template. We have developed a procedure, termed anchored PCR, whereby nucleotide sequence alterations in the template can be directly related to the quantity of amplified product. Genetic variation in the human immunodeficiency virus HIV-1 has been studied using anchored PCR. In four field isolates of the virus, the 3′LTR was compared both by PCR analysis of DNA from virus cultures and DNA sequencing. DNA templates that matched the primers varied less than threefold in PCR product yield, whereas significant 3′end primer-template mispairing decreased PCR product 10- to 100-fold. Using these guidelines for genetic variability manifested through PCR, 40 PCR primers encompassing the GAG, ENV, and 3′LTR segments of the genome were used to compare sequential HIV-1 isolates from six patients. Some primers were apparently located in genomic regions without significant interisolate variability, as they yielded equivalent amounts of amplified DNA from all the isolates. The quantity of amplified DNA obtained with other primers varied 10- to 100-fold among patients, but was consistent for sequential isolates from an individual patient. Two African HIV-1isolates were readily distinguished from a panel of North American isolates by the same method. Systematic classification of HIV-1 genetic variants may be possible by anchored PCR.
AB - Summary: Amplification of DNA by polymerase chain reaction (PCR) is influenced by the homology of oligonucleotide primers with the DNA template. We have developed a procedure, termed anchored PCR, whereby nucleotide sequence alterations in the template can be directly related to the quantity of amplified product. Genetic variation in the human immunodeficiency virus HIV-1 has been studied using anchored PCR. In four field isolates of the virus, the 3′LTR was compared both by PCR analysis of DNA from virus cultures and DNA sequencing. DNA templates that matched the primers varied less than threefold in PCR product yield, whereas significant 3′end primer-template mispairing decreased PCR product 10- to 100-fold. Using these guidelines for genetic variability manifested through PCR, 40 PCR primers encompassing the GAG, ENV, and 3′LTR segments of the genome were used to compare sequential HIV-1 isolates from six patients. Some primers were apparently located in genomic regions without significant interisolate variability, as they yielded equivalent amounts of amplified DNA from all the isolates. The quantity of amplified DNA obtained with other primers varied 10- to 100-fold among patients, but was consistent for sequential isolates from an individual patient. Two African HIV-1isolates were readily distinguished from a panel of North American isolates by the same method. Systematic classification of HIV-1 genetic variants may be possible by anchored PCR.
KW - Genetic variation
KW - HIV-1
KW - PCR typing
UR - http://www.scopus.com/inward/record.url?scp=0025836730&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0025836730&partnerID=8YFLogxK
M3 - Article
C2 - 1941529
AN - SCOPUS:0025836730
VL - 4
SP - 1241
EP - 1250
JO - Journal of Acquired Immune Deficiency Syndromes
JF - Journal of Acquired Immune Deficiency Syndromes
SN - 1525-4135
IS - 12
ER -