Physiology 3140A Study Guide - Quiz Guide: Amplicon, Ethidium Bromide, Agarose Gel Electrophoresis
22 May 2018
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Physiology 3140
PCR Module
Polymerase Chain Reaction (PCR)
- Amplifying DNA
- Done by adding an enzyme (DNA dependent DNA polymerase) that makes a complimentary
copy of a template strand
- Reaction temperature is increased in vitro to separate the double stranded DNA
- Primers are added to the mixture of DNA that are specific to the segment of DNA that needs to
be amplified at the 3 end
- The primers will anneal to each strand
o One primer is called the forward aka 5 primer
o The other primer is called the reverse aka 3 primer
- Nucleotides (dNTPs) are added into the reaction tube so the polymerase has access to the
building blocks to extend the sequence its copying
- The polymerase in rxn is initially inactivated bc the rxn needs a huge increase in temperature
- Thermal polymerase that dont denature in higher temperatures and so dont need to be
replaced at the end of each cycle of DNA replication
- Cycle components:
o Denaturation: separates the strands of DNA (94-100 degrees C)
o Annealing: allows for primers to bind to DNA (variable based on the sequence of the
primers ) (50-65 degrees C)
o Elongation: optimal temperature for the polymerase to function (70-75 degrees C)
- VIDEO:
o PCR is a method to make many copies of a segment of DNA starting with a very small
amount
o This technique can be used to identify specific microorganisms from a small amount of
DNA nad to identify persons involved in crimes in DNA on cigarettes or hair strands
o The DNA to be amplified is mixed with dNTPs, taq polymerase (thermal stable) and DNA
primers
o The DNA primers hybridize to the ends of the gene to be amplified and provide a
starting point for the taq polymerase
o The mixture is heated to break the H bonds in the DNA, forming single stranded
molecules
o The mixture is then cooled to allow the DNA primers to anneal to each end of the
segment to be copied
o Taq polymerase then creates the complementary strand of DNA using the primers as the
starting point
o Temp is raised again to separate the DNA strands and then lowered again to allow the
primers to attach
o Taq poly now creates another set of complementary strands
o This is repeated until enough DNA is created to be identified or used for further
research
o After 21 cycles, one molecule of DNA can be amplified to over 1000 000 copies
Visualizing a PCR
- The amplified products are separated and visualized on an agarose gel
- The gel contains:
o Ethidium bromide
o Intercalating agent that makes double stranded DNA visible under UV light if there is a
sufficient number of DNA copies
find more resources at oneclass.com
find more resources at oneclass.com
- Amplicon= the amplified product(s)
- The amplicon should be a predicted size based on the primers that were added
- There is a databse available to design primers and predict the final amplicon size
- The amplified DNA that is run on the gel should be this predicted size
- BUT, for paternity testing or identification of the owner of a DNA sample found at a crime scene
specific primers are designed that would create variable amplicon sizes depending on the owner
of that DNA
o Ex: in multiple ppl, a segment of DNA may produce an amplicon that is 1000 bp in length
but in other people (due to genetic variability) that same region of the genome produces
an amplicon that is 500 bp in length
o Primer design to identify (creating a DNA finger print) a person of interest relies on the
creation of variable sized amplicons
o SO, primer design will be based on whether variation is desirable to similarity is
desirable
o Usually, the differences in primer design for these 2 parameters would amplify a region
of DNA that does not encode a gene (variable amplicon produced) or with the exon of a
gene (amplicon of the same size)
- Paternity testing example:
o The primers used here are called variable number of tandem repeats (VNTRs)
o These are heritable and children will only contain these in their genome if it has come
from their parents
o Here, 2 children are from the current marriage, one child is from a previous marriage
and one child is adopted
o Daughter 1 has amplified 2 VNTRs from her mother and 2 from her father (confirming
paternity)
o Son 1 also amplifies 2 VNTRs from the mother and 3 from the father
o D2 does not amplify VNTRs from this father but VNTRs from her mother →showing a
different father not run in this study (child from previous marriage)
o Son 2 does not amplify VNTRs from mother or father which in this study was not
surprising bc he is adopted
Reverse Transcriptase (RT-PCR)
- Using PCR to identify the small amoutns of mRNA transcripts that are present within a cell to
answer questions about levels of gene transcription
- Since PCR relies on a DNA template, the mRNA needs to be converted to DNA
- mRNA is first converted into a single stranded DNA using the mRNA as a template, followed by a
copying the single DNA stranded into a double stranded sequence for subsequent PCR
- the resulting DNA is called cDNA
- in reverse transcription, a polymerase is used that matches nucleotides to the template strand
but the polymerase is an RNA dependent DNA polymerase (a type of reverse transcriptase
enzyme)
- a primer must also be used to allow for the polymerase to extend the sequence to make a copy of
cDNA
- now, the cDNA can be used in a regular PCR to amplify the sequence
- VIDEO:
o Reverse transcription is the process of making complementary DNA from single
stranded RNA
o Requires:
▪ RNA
▪ Reverse transcriptase enzyme
▪ Primer
▪ dNTPs
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Done by adding an enzyme (dna dependent dna polymerase) that makes a complimentary copy of a template strand. The primers will anneal to each strand. Nucleotides (dntps) are added into the reaction tube so the polymerase has access to the building blocks to extend the sequence its copying. Reaction temperature is increased in vitro to separate the double stranded dna. Thermal polymerase that don(cid:495)t denature in higher temperatures and so don(cid:495)t need to be. The polymerase in rxn is initially inactivated bc the rxn needs a huge increase in temperature replaced at the end of each cycle of dna replication. Video: pcr is a method to make many copies of a segment of dna starting with a very small amount, this technique can be used to identify specific microorganisms from a small amount of. The amplified products are separated and visualized on an agarose gel.
