PCR Steps

• In DNA replication, it requires existing double-stranded DNA to be the TEMPLATE (to give the order of nucleotide bases) in the form of?

  Deoxynucleotide triphosphates
• What are the 4 deoxynucleotide triphosphates?

  dATP

  dGTP

  dCTP

  dTTP
• What catalizes the addition of nucleotides to the growing STRAND?

  DNA Polymerase
• It is synthesized by a primase (in vivo) to which DNA polymerase adds subsequent bases.

  Primer
• PCR can produce millions of copies-amplicons of DNA in how many hour(s)?

  Less than 2 hours
• The DNA template, short oligonucleotide primers, nucleotides, polymerase, and buffers are subjected to a/an?

  Amplification Program
• An amplification program consists of a specified number of cycles that are divided into steps in which samples are held at a particulat temperature for designated times, the steps of a PCR cyle are?

  Denaturation

  Annealing

  Extension
• Temperature of Denaturation?

  90-96 degrees celcius
• Temperature of Annealing?

  50-70 degrees celcius
• Temperature of Extension?

  68-75 degrees celcius
• Denaturation time?

  20-60 seconds
• Annealing time?

  20-90 seconds
• Extension time?

  10-60 seconds
• It is a step in PCR wherein double-stranded DNA is separated into 2 single strands?

  Denaturation
• It is a PCR step wherein two oligonucleotide primers hybridize to their complementary DNA sequences and the most critical step for specificity?

  Annealing
• They dictate the part of the template that will be amplified and determine the specificity of the amplication.

  Primers
• Startimg point is determined using the?

  Tm or melting point
• What is the shortened formula of melting temperature?


  Tm=81.5 degrees celcius+0.41(%GC)-(675/n)
• It is at which 1/2 of the double-stranded DNA exist as a single-stranded DNA?

  Temperature
• Melting Temperature is affected by?

  Reaction Conditions

  Salt Concentration

  Mismatches

  Template Condition

  Secondary Structure
• It is a step in PCR where DNA synthesis occurs and it is performed by DNA polymerase?

  Extension
• It synthesizes a copy of template DNA?

  Polymerase
• DNA polymerase calalyzes the formation of the phosphodiester bond between an incoming dNTP, determined by hydrogen bonding to the template (A:T or G:C) and the base at the 3’ end of the primer.
• End result of Extension.

  2x template number