Nucleic acid probes and PCR

sugar and phosphate

what comprises the DNA backbone?

backbone

where are nucleic acids attached to?

• adenine

• guanine

• cytosine

• thymine

what are the nucleic acids in DNA?

• adenine

• guanine

• cytosine

• uracil

what are the nucleic acids in RNA?

• A-T

• G-C

what are the nucleotide pairs in DNA?

• A-U

• C-G

what are the nucleotide pairs in RNA?

re-annealing

what does denaturation mean?

technology based on identification of genes of DNA sequences

what is the nucleic acid probe method?

gene probe

what is another name for the nucleic acid probe method?

specific

what is the specificity of the labeled piece of DNA/RNA in the nucleic acid probe method?

identifies complementary strand of DNA in patient specimen

what is another function of the nucleic acid probe method?

the binding of two complementary strands

what is the hybridization procedure of the nucleic acid probe method?

• radioisotopes

• enzymes

• fluorochromes

• chemiluminescent

what are some examples of the variety of labels used in the nucleic acid probe method?

labeled probe is mixed with sample and the mixture is manipulated and tested for the attached probe

what is the procedure of the nucleic acid probe method?

a single strand of unique DNA prepared through molecular techniques and label

what is the probe used with the nucleic acid probe method?

material present in sample

what is the target in the nucleic acid probe method?

cells from specimen affixed to filter→lyse cells and generate single-stranded target DNA→add reporter-labeled probe; allow for reannealing to target→measure hybridization directly if reporter is radioactive or fluorescent. add enzyme substrate if reporter is an enzyme→detection

what are the steps to the nucleic acid probe method?

increases the number of DNA targets by repeatedly copying a specific DNA sequence

what is the principle of Polymerase Chain Reaction (PCR)?

• template DNA

• DNA polymerase

• Primers

• Nucleotide bases

what are the components of PCR?

DNA to be amplified

what is template DNA?

the enzyme that makes DNA

what is DNA polymerase?

Taq polymerase

which DNA polymerase typically is used in PCR?

short pieces of DNA that bind to a specific region of template DNA

what are DNA primers?

adenine, guanine, cytosine, and thymine

what nucleotide bases are used in PCR?

• denaturation

• annealing

• extension

what are the steps to PCR?

the separation template DNA into two strands

what is denaturation?

a step in which the reaction mix is cooled to allow primers to anneal to the target sequence

what is annealing?

the primers initiate DNA synthesis using DNA polymerase

what is extension?

• millions of copies of target DNA

• amplified DNA molecules

• detected using different methods: agarose gel electrophoresis, nucleic acid probe, etc

what are the results of PCR?

amplicon

what are amplified DNA molecules called?

• microorganisms and viruses that are difficult to grow

• identification of human leukocyte antigens for tissue transplantation

• measurement of cytokine activity

• reverse transcriptase PCR

what are the applications for PCR?

gold standard for detection and monitoring therapy in hepatitis infections, and used for HIV detection and monitoring

what is reverse transcriptase PCR?

plasma with HIV→lyse HIV→RNA genome of HIV→reverse transcribe→DNA copy of HIV genome→PCR→multiple DNA copies of HIV genome→assay

what are the steps of reverse transcriptase PCR?

lower CD4 and higher RNA count

what does a poor prognosis of PCR look like?

higher CD4 and lower RNA count

what does a good prognosis of PCR look like?

• conditions must be rigidly controlled

  • clean areas and procedures in place to avoid contamination

• false positive may occur if sample, reagents, or laboratory equipment contaminated

what are some sources of error with PCR?

gives quantitative results by monitoring DNA synthesis during PCR

what is the function of real-time PCR (qPCR)?

fluorescence increasing as the dye binds to the increasing amount of DNA

what does qPCR measure?

no post-PCR processing

what is different between PCR and qPCR?