PCR Digital Lab – Jason Bao

Day 1
Go to the following website for the PCR Digital Lab: https://learn.genetics.utah.edu/content/labs/pcr/
1) What does PCR stand for? Why would it have this name?
PCR stands for Polymerase Chain Reaction. It has this name because it can generate 100 billion identical copies of a
specific DNA sequence in a matter of hours.
2) What are samples that you can use in PCR?
To perform a PCR reaction, a scientist needs DNA that has been extracted from cells. For example, the scientist can
extract DNA from a small sample of blood, skin, saliva, or hair follicles.
3) What is the special design feature of PCR tubes?
PCR tubes are specially designed for even heat distribution as PCR works by heating and cooling the solution over and
over.
4) Why are your solutions in ice buckets? I know the lab doesn’t say the answer, so THINK!
The solutions are in ice buckets to protect the samples and prevent the solutions from possibly degrading at these warmer
temperatures as at the low temperature of the ice buckets, the reaction rate is much slower as proteins like enzymes are
much less efficient, which decreases the possibility to degradation.
5) Why are primers added to the tube? What would occur if you didn’t add them?
Primers are added to the tube to attach to the sites that are at wither end of the segment that the scientists wants to copy.
Because there is almost no chance these primers will target the wrong sites, they are an important tool for copying very
specific DNA sequences. If primers were not added, then DNA replication will not occur (no product).
6) What must you add after the primers? What would occur if you didn’t add them?
Nucleotides must be added to the PCR tube right after the primers. If the nucleotides (A,C,G,T) are not added, the primers
will not have the genetic building blocks used to create billions of DNA copies, so replication cannot occur.
7) What is the third thing that must be added into your PCR tube? What would occur if you added all previous
solutions but forgot this one?
The third thing that must be added into the PCR tube is the enzyme Taq Polymerase (DNA Polymerase) which will read
the DNA code and attach the matching nucleotides to create DNA copies. If Taq Polymerase is not added, there will be no
enzyme to creating a matching strand of DNA so replication can likewise not occur.
8) What is special about the final substance you must add to the PCR tube?
The DNA Polymerase (Taq) added is special as it is specially selected to withstand the high heat of the PCR reaction.
9) What is the PCR machine called? Why is it called this?
The PCR machine is called the DNA Thermal Cycler. It is called this as this machine can precisely heat and cool the PCR
tube at specific times across an hour, temperature changes crucial for the success of the reaction.
10) What occurs when the temperature
a. Drops to 50 degrees C?
Single-stranded DNA molecules naturally attempt to pair up. However, because there are many more primer sequences
than DNA strands in the tube, the primers crows their way in and lock onto their target before the DNA strands can rejoin.
 b. Rises to 72 degrees C?
At 72 degrees Celsius, Taq Polymerase is activated. It locates each primase attached to a single DNA strand and begins
adding complementary nucleotides onto the strand. Taq Polymerase continues to add these nucleotides until it reaches the
end of the strand and falls off.
c. 95 degrees C?
The increase in the temperature causes the DNA strands to separate again, allowing the cycle to repeat itself.
11) What is the result of Cycle 3?
During cycle 3, the scientist’s desired products begin to appear – two strands that begin with primer one and end with
primer two. There are DNA copies of the segment of DNA that the scientists was targeting. As such, two desired
fragments are produced, and as the cycles progress, these desired fragments will become a majority.
12) How many cycles would typically occur to give a good result? Why?
30 cycles would need to typically occur to give a good result as after thirty cycles, there are over a bullion target sequence
fragments and only sixty copies of the longer lengths of molecules. As such, a scientist would have a solution of nearly
pure target sequence.
13) What is the point of doing PCR? How can the results be used?
The purpose of PCR is to amplify and replicate small segments of a target DNA region so that it can be analyzed or used
in some way by the scientist. Additionally, because PCR is a relatively fast and inexpensive technique to perform this
amplification, it is popularly used. The results of PCR can be used in numerous settings ranging from forensics to genetic
testing. In forensics, a scientist can use PCR to amplify a DNA sample of hair to match the genetic marker with potential
suspects while in genetic testing, a scientist can amplify genes associated with genetic disorders from patient DNA in
order to further and more closely analyze them.
Day 2
Go to the following website and read: https://www.addgene.org/protocols/primer-design/ This includes watching the
video at the bottom.
1) What are PCR primers? How many are needed? Why?
PCR primers are short, single-stranded DNA sequences that are complementary to the template region of DNA, anneal to
the regions upstream and downstream of the DNA segment to be amplified, and usually have a guanine or cytosine at the
3’ end. Essentially, they provide a 3-OH group for Taq polymerase to add nucleotides to.
Two PCR primers are used in each PCR reaction, a forward and reverse primer, to flank the target region for
amplification. Because these primers have sequences that will make them bind to opposite strands of the template DNA,
two new copies can be synthesized per cycle, enabling the exponential amplification of the target DNA.
2) How can you get PCR primers?
To design primers, a scientist must limit their sizes to a length of 18-24 bases, an approximate 40-60% G/C content, start
and end the primers with 1-2 G/C pairs, ensure a melting temperature of 50-60 degrees C, and verify that the primer pairs
have a melting temperature within 5 degrees C of each other and that they do not have complementary regions.
3) What is the difference between the forward and reverse primers?
The forward primer binds to the template DNA while the reverse primer binds to the other complementary strand, which
allows both strands to be amplified in the PCR reaction.
4) What would happen if you forget to add the PCR primers to your PCR mix prior to inserting them into the
machine? Support your claim.
If a scientist forgets to add the PCR primers to the PCR mix prior to insertion into the machine, the
amplification/replication reaction would fail as Taq Polymerase (DNA Polymerase) cannot add nucleotide bases without a
preexisting 3’-OH group from a small piece of DNA that the primer provides (Taq Polymerase needs a primer to add the
first nucleotide).
Now it’s time to be a scientist. This is a website that we can use to design primers for real life experiments.
https://www.ncbi.nlm.nih.gov/tools/primer-blast/ Look around. Click on the “?” buttons for better explanations.
5) Now, pick a gene of interest on the human genome. Yep, pick your choice of gene. It will have a letter and
usually number sequence.
TYR gene – skin color because codes for tyrosinase:
1 mltdaryddf vwthieqtls ihgttnflsw hryftwtyeq alrdecgyrg yqpnwgktal
61 dpvnsqvfdg spysmggngd yvphnctnal psglncipag ngggcvtqgp frdmkvnlgp
121 vtptlaaegi vssspvsayn prclrrdits wvssrwstda isaslirend distfqtvmq
181 gdfasgfygv htaghftmag dpsgdifasp gdpafwlhha qidrtwwiwq nqnlaarqna
241 iggtitldns pasrpgrled plslgvnapn itigdamstl agpfcyiyl

6) Use Primer BLAST to create primers for your gene of interest. Write down what you did to get your results.
In order to get my results, I typed the accession sequence in the PCR Template tab and set my range for both the forward
and reverse primer from 5’ to 3’, the PCR product size from 70-100, number of primers to return to 10, and primer
melting temperatures from 57.0 to 63.0 degrees Celsius with 60.0 being the optimal temperature. Then, I used the
automatic/given choices for the exon/intron selection and primer pair specificity checking parameters as the given choices
already best suited the primer I was creating. After all of this, I clicked get primers and the Primer BLAST generated a
detailed primer report for my use.
7) Now RESEARCH! Where can you buy/order primers from? How much does it cost? Can you design your own?
You can buy/order PCR Primers (oligonucleotides) from places like Thermo Fisher Scientific and Integrated DNA
Technologies. At Thermo Fisher Scientific, you can buy custom standard DNA oligos for $2.66 each while at Integrated
DNA Technologies, you can buy special rhPCR Primers with customizable bases form $25.00.
Yes, one can design his or her own primers through these companies as they allow customers to choose the oligo sequence
they would like.
Finally, read Tip 2 and 3 on this website: https://bitesizebio.com/9951/three-tips-and-two-tricks-for-using-blast/
8) In your own words (and most likely a paragraph) explain what tips 2 and 3 are saying.
First, tip two discusses the premise of effectively using NCBI’s Primer-BLAST to design primers. Before filling out any
of the other boxes of the Primer-BLAST, it is important to enter a target sequence accurately buy cutting or pasting or as
an accession number. Then, it highlights key parts of the next parameters that Primer-BLAST, noting that range will
normally be numbered 5’ to 3’, instructing users to choose “use my own forward primer” if they’ve already designed their
primers, and clarifying that PCR product size is the range of acceptable lengths of the PCR products. Finally, tip two
elaborates on the last couple questions Primer-BLAST asks, clarifying that # of primers to return is the preferred number
of candidate sets of primers to consider, primer melting temperatures lets one specify Tm, exon junction-junction is
marked if one wants to exclude genomic DNA, generally leave specificity checked unless one wants Primer-BLAST to
return off-target primers, and finally noting that splice variant handling only applies to mRNA sequences.
Next, tip three explains how to check if any primers hit anything off-target. First, in the query box of Primer-BLAST,
enter the forward primer and type in 20 N’s in a row to separate the primers into non-overlapping alignments. Then,
specify the database that the primers are blasted against such as a human gene and DNA. Finally, once the results are
generated, the scientist should check for certain combinations.
Day 3
Today it is error day! Research 15 common errors made in PCR. List them and then in a sentence or less explain how/why
that error causes the results it does.
15 common errors:
1. Poor RNA Quality – Because RNA is extremely sensitive to degradation by RNases, degraded or impure RNA
can limit the efficiency of the RT and may not give an accurate representation of gene expression.
2. Not using “master mixes” – A master mix, essentially a mixture of reaction reagents, is used to minimize
variability between samples and therefore improve reproducibility, so if it is not used, errors can be greatly
amplified.
3. Cross-Contamination – If all surfaces in the PCR area are not routinely decontaminated, the PCR data will be
distorted and show unexpected amplicons that contaminate the finished PCR product.
4. Not using a “-RT” control – If a minus-reverse transcriptase control is not used, genomic DNA will not be
completely eliminated from RNA preparation, which contaminates RNA preparation and distorts the final data.
5. Poor primer and probe design – Without the proper design of primers and probes such as with a primer design
software program, the amplification can be unsuccessful & sporadic, potentially contaminating the genomic
DNA.
6. Using an inappropriate normalization control – An invariant endogenous control is often used in the assay to
correct for sample to sample variations in PCR efficiency and errors in sample quantitation, so failing to use the
appropriate one can decrease the reliability of the PCR experiment.
7. Melting curves are not performed when using SYBR Green – SYBR Green I fluorescence is a direct measure of
accumulation of the product of interest so if it is not used, the dissociation curve will reveal a series of peaks
instead of one sharp peak at the melting temperature that prevents the scientist from successfully discriminating
between specific and non-specific reaction products.
8. Not setting the baseline and threshold properly – If the baseline is not set two cycles earlier than the Cq value for
the most abundant sample, the scientist will not obtain accurate Cq values and the resulting data would not be
meaningful.
9. Poor reaction efficiency – Changes in the length of the amplicon, secondary structure, and primer design from the
optimal ranges/conditions can negatively affect the efficiency of the reaction.
10. Using an inappropriate range for standard curves – If the range of a standard curve is not set to extend above and
below the expected abundance of a scientist’s target, then the scientist will inadvertently miss necessary data.
11. Lack of organization – A lack of organization around the scientist’s table can cause immense confusion as to what
has been pipetted and what has not and can lead to the mix-up of primers that leads to the overall failure of the
PCR.
12. Skimping on controls – Since negative controls (“no template controls”) can be used to detect cross contamination
of surfaces or reagents, by skimping on these controls, a scientist cannot detect DNA contamination in his or her
sample, which limits the confidence in his or her findings.
13. Copying errors – Editing errors that occur during DNA polymerase-catalyzed enzymatic copying leads to the
amplification of DNA by PCR with many errors.
14. DNA thermal damage – DNA thermal damage can lead to cytosine deamination and depurination that will lead to
an accumulation of numerous errors during the PCR amplification process.
15. Forgetting to add primers, nucleotides, or Taq polymerase – If any of these crucial inputs are not added to the
PCR tubes, then the reaction will fail as the necessary compounds are not present to be used to amplify the target
DNA.
16. PCR machine thermal block is malfunctioning – If the thermal block, the inner tube holder which generates heat,
of the PCR machine is faulty, Taq polymerase cannot be activated and the DNA strands cannot be separated,
causing the PCR reaction to fail.
17. The DNA polymerase enzyme has stopped working – If the DNA polymerase enzyme either stops working or is
no longer as efficient, perhaps due to freeze-thawing, then the extension step during the PCR reaction will be
incomplete and no PCR product is created.
18. Unsuitability of PCR primers – Sometimes, despite planning, originally designed PCR primers will not work in
the real world, which would prevent Taq polymerase from starting to amplify the DNA.