BSC LAB 7

OSCEOLA is set up to run how many tests per day

+1,000

mixtures to be separated by gel electrophoresis are usually

- nucleic acids of different sizes (DNA or RNA with a negative charge) - proteins of different sizes and charges

makes the PCR technique more convenient and time effective

- thermocycler no longer need to: - have multiple water baths at different temps. - move sample between water baths - add enzyme several times

tandem repeat regions can be used to compare individuals by

- using multiple repeat sites - using multiple primers

body fluids can contain 2 types of pathogens

-blood-borne -saliva-borne

OSCEOLA allows

-high-throughput testing - many (88) samples to be tested at once

end product of PCR results in how many copies of target DNA

2

the PCR cycle has how many steps, what are they called.

3 - DNA Denaturation - Annealing - Extension

__ assays target the virus genome, ___ human control

3, +1

how many target cycles in PCR

30

at the end of 25 cycles, approx. how many copies of the target DNA will there be?

33 million

many (88) samples can be tested at once, using __ assays for each sample

4

Taq Polymerase functions optimally at

72 C

What is the purpose of including a PCR reaction with no added DNA? Isn't this just a waste of expensive enzyme when we know that without template, you will not get any PCR products?

A PCR reaction with no added DNA is used as a negative control. This helps to rule out any false positive results during the amplification process. A experiment is considered flawed if amplification is observed in the negative control.

difference between genes and alleles

A gene is a specific section of a chromosome where the base pairs that code for a specific characteristic are stored, whereas an allele is the sequence of the base pairs in the section. For example, a gene is responsible for the trait of hair color, and there is an allele for blonde hair, brown hair, red hair, etc. Essentially, alleles are different versions of a gene.

DNA fragment

BAND

on a gel, __________ will show the different sized fragments

BAND

What is the purpose of using DNA polymerase from Thermus aquaticus for the polymerase chain reaction rather than a DNA polymerase from a better characterized bacterium such as E. coli?

Because DNA Polymerase from Thermus aquaticus can withstand higher temperatures than E. coli. DNA Polymerase from Thermus aquaticus can cycle through many temperature shifts without being denatured, preventing the need for a fresh enzyme before every cycle.

split the double helix into two single strands

DNA denaturation

sequence

DNA extraction -> PCR -> Gel Electrophoresis

known sizes of DNA for reference

DNA markers AKA Ladder (Standards)

Describe how gel electrophoresis is used to separate molecules and identify their approximate sizes.

Gel electrophoresis separates molecules according to their size and shape by forcing them to migrate in an electric current through a semi-solid matrix. Take, for example, a solution of DNA molecules placed in a gel. Because each DNA molecule is negatively charged, it is pulled through the matrix by the electric current. Note that small molecules move more quickly than larger ones. The smaller molecules are the molecules (bands) that are furthest from the start of the gel (matrix), whereas the larger molecules are the molecules (bands) closest to the start of the gel (matrix).

starting material for PCR

Genomic DNA (sample of chromosomal DNA)

PCR was developed by ____________ in 1983

Kary Mullis

the path the DNA travels

LANE

a method of PCR COVID-19 test modified by Dr. Jonathan Dennis that allows high through-put testing

OSCEOLA

amplifies a target DNA sequence within a larger population of DNA by artificial replication (in vitro); up to a million-fold amplification of target DNA

PCR

lasts for several hours

PCR

what COVID test is most reliable

PCR (molecular test)

detects a piece of the virus genome and indicates current infection.

PCR AKA 'molecular test' - most reliable

two types of current infection tests

PCR, antigen

different types of COVID tests

PCR, antigen, antibody

heat-stable enzyme that can cycle through many temperature shifts without being denatured, thus preventing the need for fresh enzyme before every cycle

Taq Polymerase

specialized primers and devices used to detect PCR products

TaqMan Probes

Taq polymerase name is derived from the name of this bacterium

Thermus Aquaticus

thermophilic bacterium found in hot vents

Thermus Aquaticus

The polymerase chain reaction can only be used to amplify genes that have already been cloned and sequenced. Why is this true?

This is true because, in order to amply a segment of a gene using PCR, you first need the primers of that gene. Primers are single-stranded oligonucleotides that are complementary to the flanking sequence. Primers can only be designed when the flanking sequence on either side of the target DNA is known.

(T/F) OSCEOLA is partnering with TMH, providing medical technicians to perform tests

True

(T/F) there are no known incidents involving the transmission of blood-borne pathogens by saliva (when no blood is present)

True

comb impression to load sample into

WELL

If you started a PCR protocol with 10 double-stranded molecules of DNA, how many double-stranded molecules of product would you have after 5 cycles of PCR? Explain your reasoning.

You would have 320 double-stranded molecules after five cycles of PCR. Starting with 10 double strands.... Cycle 1 = 20 Cycle 2 = 40 Cycle 3 = 80 Cycle 4 = 160 Cycle 5 = 320

polymers used in the gel matrix

agar, agarose, or acrylamide

derived from seaweed - used for gel electrophoresis in this lab

agarose

different versions of a gene

alleles

OSCEOLA is not held back by reagent availability because it uses

alternative reagents/materials to the pre-made test kits

primers attach to flanking sequences at the 3' end

annealing

detects antibodies (proteins in blood)

antibody

detects protein on/within the virus

antigen

Each cycle of the polymerase chain reaction consists of a shift between three different temperatures (in our protocol, 94°C, 60°C, and 72°C). In your own words, explain what occurs at each of those temperatures.

at 94 degrees Celsius, DNA Denaturation occurs. This is when the double helix of the DNA molecule is split into two single strands. This step usually takes approximately 30 seconds. At 60 degrees Celsius, the Annealing process occurs. DNA primers attach (or anneal) to the "template" DNA. This process takes approximately 30 seconds. Finally, at 72 degrees Celsius, the DNA Polymerase enzyme extends the primers, making a new strand of DNA. This process is called Extending, and takes approximately 1 minute.

DNA samples can be collected from a wide range of sources, in humans it can be collected from:

blood, hair, saliva

can be used to determine parentage

comparing the similarity in tandem repeat regions

driving force of electrophoresis

electric current

DNA polymerase enzyme extends the primers (5' to 3')

extension

this technique separates molecules according to their size and shape by forcing them to migrate (1) in an electric current (the driving force) (2) through a semi-solid matrix (the gel)

gel electrophoresis

contain microscopic pores of known approximate sizes

gels

discrete units of heredity information located on a chromosome

genes

what bonds are broken during the denaturation step of the PCR cycle

hydrogen bonds

what does detecting three different parts of the virus genome do to the test

increases reliability of the test

short tandem repeats are used for certain DNA tests, these patterns can help determine an individuals

inherited traits

the distance migrated through the gel is

inversely proportional to the size of the DNA molecule

the larger, longer molecules (more based pairs) move

more slowly through the gel matrix - the distance they travel is very short

denaturation and extension temperatures depend on the

polymerase being used

used to "amplify" or "increase the quantity", a part of the DNA; is one of the most commonly used techniques in molecular biology

polymerase chain reaction (PCR)

are 20 nucleotides long and are complementary in sequence to the target DNA

primers

are single stranded oligonucleotides complimentary to the flanking sequences

primers

used for the replication of the target DNA

primers

annealing temperature depends on the

primers being used

occurs in DNA when a pattern of nucleotides is repeated, and the repetitions are directly next to each other on the chromosome

tandem repeats

DNA sample of interest

target DNA

"ingredients" needed for PCR

target DNA, primer, Taq Polymerase, free nucleotides

during PCR each step of the cycle takes place at a different

temperature - DNA Denaturation (94 C/30 sec) - Annealing (60 C/30 sec) - Extension (72 C/ 1 min)

must be known on either side of the target DNA so that primers can be made

the flanking sequence

the smallest, most tightly packed molecules travel

the greatest distances - they can be "pulled" through the pore spaces in the gel matrix by the current much easier

the fragments in the Tandem Repeat Regions will be different sizes for each of the sites/primers, depending on

the number of repeats

varies with the polymerase being used and the size of the DNA fragment being amplified

time of each step - extension is approx 1 min/1,000bp using Taq polymerase

the type (DNA, RNA, protein) and size (MW) of molecules to be separated will determine the

type of gel used