Chapter 7 Flashcards
Amplification
single stranded 18 to 30 bases in length DNA fragments complementary to sequences flanking the region to be Amplified
PCR primers
what do primers do
determines specificity, and the distance determine size of product
technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence
polymerase Chain Reaction PCR
PCR products that are just double the size of the primers. Result from binding of primers on to each other through short homologies after three prime ends and copying each primer sequence
primer dimers
design to add or alter sequences to one or both ends of the PCR product
tailed primers
heat stable polymerase isolated from thermus aquaticus
Taq polymerase
taq polymerase lacking 289 n terminal amino acids
stoffel fragment
product of PCR reaction
amplicon
making copies of a Target sequence to such a level that they can be detected in vitro
amplification
short oligonucleotide primers, nucleotides, polymerase, buffers
components of PCR DNA template
what are the three cycles of PCR
denaturation, annealing, extension
fluorescent detectors measure PCR product as the reaction proceeds
real-time PCR
small sample volumes in Chambers that can be heated and cooled quickly by changing the air temperature surrounding sample
rapid PCR
withstand the repeated high denaturation temperature
thermostable polymerase
changes temp in a block or chamber holding the sample
thermal cycler
what PCR cycle is where double-stranded DNA is denatured into two single strands by heating the sample at 90 - 96 degrees C for 20 minutes
denaturation
what PCR cycle is the most critical step where primers determine the specificity of the amplification at 50-70 degrees C for 20 seconds
annealing
what PCR cycle is where DNA is synthesized at 68 - 75 C for 30 seconds
extension
what may occur due to non-specific hybridization of primers
Misprime’s
isothermal, probe amplification, probes bind immediately adjacent to one another on template, ligated and become templates for The Binding of more probes. For chlamydia gonorrhea and sickle cell
ligase chain reaction
isothermal, signal amplification, series of hybridisations attaches multiple signals to each Target molecule. HPV, HCV, HIV 1
branched DNA
isothermal, signal amplification, immobilized DNA probe binds to RNA targets. RNA DNA hybrids are bound by LABELED monoclonal antibodies. HPV, HBV, CMV
hybrid capture
isothermal, Target amplification as RNA, cDNA is made from RNA Target adding RNA polymerase promoter, RNA synthesis from cDNA template and can serve as a source of new cDNA. Tuberculosis, chlamydia, HIV, CMV
TXN- mediated amplification (TMA)
two probes, one has donor five prime one has acceptor 3 Prime Vines two adjacent Target’s Fluoroscein-rhodamine
FRET
Quencher and reporter together, broken when amplification occurs signal on
TaqMan
binds minor groove of double-stranded DNA
SyBr Green
what is the purpose of an amplification control in PCR
to distinguish true negatives from false negatives
what is the most dangerous contaminant in PCR
carryover PCR product from previous reaction
what is a way to avoid Misprimes, use the sequestered enzyme, keep everything on Ice, use a preheated cycler
Hot Start
uses RNA as a Target. DNA is synthesized from Target RNA then transcription of DNA produces copies of RNA
TAS transcription based amplification systems
measures the accumulation of product at annealing step in PCR cycle
molecular beacons
bind fluoresce double-stranded DNA
DNA specific dyes
bind and fluoresce intended PCR product
hybridization probes
label the PCR product
primer Incorporated probes
amplifying multiple Targets on the same strand of DNA at the same time
Multiplex PCR
what control is run to detect contamination in PCR
reagent blank
nonspecific extra PCR products can result from
Mispriming
method for purifying PCR product
put the reaction mix through a spin column
what enzyme system is used to avoid contamination in real-time PCR
dUTP - UNG
sybr green fluorescence is detectable at which stage of the PCR amplification process
primer annealing and extension
what PCR controls ensures that the enzyme is active, the buffer is optimal, and the primers are priming the correct Target sequence and must have a PCR product detected in order to be valid
positive control
Taq-Man, molecular beacons, and Scorpion type primers are all used in which procedure
quantitative PCR
complimentary of primer pairs at the 3’ end will result in what artifacts
primer dimers
what amplification procedure uses an RNA polymerase to generate amplicons
Q beta replicase
Target amplification
TMA - RNA is synthesized from cdna template. TB, C. trachmatis, HIV, CMV
probe amplification
ligase chain reaction - ligate adjacent primers together requires a thermal cycler to change temp. chlamydia, gonorrhea, Sickle Cell mutation
signal amplification
branched DNA - short oligomer probes used to capture Target nucleic acid. Hbv, hcv, hiv-1.
hybrid capture
detection and characterization of HPV. HPV, hbv, CMV. four recognized by antibodies. bound to single stranded RNA probes
