DNA Quantitation and PCR

What can occur if a sample contains too much DNA?

Off-scale peaks, split peaks, and locus to locus imbalance

What can occur if a sample contains too little DNA?

Locus and allele dropout. Heterozygous peak imbalance

How did UV-Vis quantification work?

Samples were analyzed with UV-Vis of 260 nm. The less light that passed through, the more highly concentrated the sample

How did Yield gels work?

DNA was run on an agarose gel and stained. The bands were compared to standards.

How was quality of extraction determined in yield gels?

Smears indicated many fagments while tighter bands were associated with better extraction

What was the first human-specific quantitation method?

Slot blot

How is DNA bound in the slot blot?

DNA is pipetted into wells then distributed across the membrane by positive charge

How was DNA detected in the slot blot?

Probe would bind specifically to human DNA. A biotin marker would then bind to the probe, followed by horseradish peroxidase which would oxidize the TMP to create blue precipitate.

What is the probe used in the SlotBlot?

D1721

What are some reasons that real time PCR may not work?

Inhibitors like heme are present, Dna is degraded, insufficient DNA quantity

What is the name of the 5' nuclease assay used in quantitation?

Taqman Assay

What ist he name of the intercalating dye method?

SYBER green

How does the Taqman process work?

A probe binds to the target containing flurescent dye and a quencher. The primers bind around the probe and target. When the primer encounters the probe it cleaves it with 5' nuclease activity. The flourescent dye will be free of the quencher, producing a signal.

What is the relationship between the signal generated and the amount of DNA in Taqman?

directly proportional

What is the cycle number and how is it used?

The cycle number is how many PCR cycles it took to reach the cycle threshold. The smaller the CT, the more DNA you had in the beginning

What information does the human autosomal large rpovide?

Used an indicator for degradation

What informations does the human autosomal small tell us?

Concentration of DNA

What does IPC stand for? What does it tell us?

Internal positive control. Should be the same Ct everytime.

What are the steps of PCR?

Denature, anneal, extend

What temperaturedoes deneature occur?

94-95 C

What temperatures does annealing occur?

55-56 C

What temperatures does extension occur?

72 C

If PCR occured 100% perfectly, how many copies would you get after 32 cycles?

1 billion

Why is PCR described as exponential?

Exponential increase occurs because each new copy will become a template in the next cycle

What are PCR products called?

Amplifiers and Amplicons

How does flourescent detection of PCR work?

At least one primer in every primer pair will have a flourescent dyes

Why might we used different flourescent dyes?

Allows us to differentiate loci of similiar sizes

What occurs in PCR if your volume is too small?

May evaporate completely during heating cycles

What happens in PCR if your sample is too large?

Heating and cooling cycles take too long

What are some of the challenges faced with primer creation?

Want to avoid species homololgy and homology to other targets. Also avoid similarity between the primers?

What is a primer dimer complex? When does it form?

Forms when there's excessive 3' complementarity, primers bind to each other instead of target

What are some of the complications of multiplex pcr?

Primers must have similar melting temperatures while nto binding to each other. Amplicons shouldn't overlap in size

What are the roles of dNTPs in PCR?

Provide the building blocks of ATGC. Must put an even amount of each one intot he picture.

What is the role of MgCl2 in PCR?

Stabilizes double stranded DNA with its positive charge and is a cofactor of Taq polymerase. Too much or too little causes poor amplification

What is TAQ Dna polymerase?

Thermostable DNA polymerase with 5'-3' exonuclease activity. Processivity of 50-60 nucleotides

What is the different between 5'-3' and 3'-5' exonuclease activity?

3'-5' exonuclease is the proofreading ability- ability to go back and correct basepairs. Taq does not have this ability

What is the purpose of AmpliTaq gold?

Since polymerases can still operate under optimal temps, this is a modification that keeps the taq polymerase inactive until heated to 95

What is a Nontemplate nucleotide addition? How do we account for it?

Refers to Taq polymerase adding an A bp that wasn't originally there. We do a final extension that shifts everything to the A+ form

What's the difference between a positive and negative control in PCR?

Positive controls monitor the effectiveness of the experiment and usually invovle and controlled sample of DNA. Negative controls contain all PCR experiments except for the DNA to see if it gets contaminated

What is the ethylene oxide treatment?

A highly toxic gas used to remove background noise.