1. The following four components must be included in PCR.

Describe the function of each of the four

components in the reaction.

a- template DNA - strand used to transcribe mRNA for protein synthesis.

b- primer DNA - starting point, required since DNA polymerases can only add new nucleotides to existing
strands of DNA.

c - nucleotides - building blocks for new DNA strands.

d - DNA polymerase - reads nucleotide on template strand, brings in complementary nucleotide,

covalently linked together. Gets rid of RNA

2. List and describe the three steps in each cycle of the PCR.

First stage - denaturation occurs, hydrogen bonds are broken between bases, strands are separated

Second stage - annealing, primers bind to the 3' end of DNA template.

Third stage - extension, Taq polymerase binds to primers and copies the DNA.

3. Why are multiple cycles of PCR carried out?

Multiple Cycles are needed in order to amplify the DNA Target to millions of copies.

4. Why are two primer DNA's needed for the PCR to work?

DNA polymerase is only able to bind and elongate from a double-stranded region of DNA. One attaches
to the top and one attaches to the bottom.

5. Why did discovery of the bacterium Thermophilus Aquaticus revolutionize the use of PCR?
Taq polymerase was isolated from Thermophilus Aquaticus which was heat resistant to denaturing
allowing DNA replication to proceed without disruptions.

6. If you start with one copy of the template DNA how many copies of the target DNA will you have
after:

a - one cycle of PCR yields two copies.

b - two cycles of PCR yields four copies.

c- three cycles of PCR yields eight copies.

7. Normally, at least 25 cycles of PCR are done prior to analysis. How many copies of target DNA will
there be (assuming 100% efficiency during each cycle)?

33,554,382

8. Why are the DNA copies produced during PCR always a specific, measurable links?

Because the bases must pair up in a specific pattern.

9. How are the products of the PCR analyzed?

- the presence or absence of the target DNA sequence

- the length of the amplified fragment

- the yield of PCR product to quantitate the relative or absolute amounts of the starting DNA

- sequence analysis, either by differential probe hybridization or by direct sequencing of the product.

The simplest and most direct methods to analyze PCR products involve gel electrophoresis.

Why is it important that each copy of a target DNA be the same length?

All DNA molecules have the same amount of charge per mass, so in gel electrophoresis they will be
separated by size only.

10. Examine Clotilda's profiles.

a - how many PCR products resulted from PCR using Clotilda's DNA and primer set one?

three

b - are the products from two reactions 2 and 3 the same as the products from reaction 1? Explain.

No, each reaction used different primer sequences.

c - what information do these three different profiles give you?

Different reactions from each sample

11. Compare Clotilda's profile with those of her ex-husband Al and their children.

a - Do Clotilda and Al have similar profiles?

No.

b - would you expect the profiles of their children to be identical to the parents, different from the
parents or a mixture of the parents? Explain.

They will be a mixture because half the DNA comes from the mother and half comes from the father.

c - would you expect the profiles of Junior and Winnie to be identical? Explain. Describe a situation in
which the PCR profiles of two individuals would be identical.

No, they will have different combinations of genes from each parent identical twins would have
identical DNA.

d Are Clotildas profiles similar to those of any of the other suspects? Explain.

There are some matching with her children.

12. Compare all the profiles.

a - do any of the profiles of the clues taken from the crime scene match Clotilda? explain.

Yes, the hair, blood lens, blood floor match.

b Do any other person from the crime scene match any of the suspects? Explain.

Yes, Spike matches with the blood handle and saliva sample.
 General Affidavit
State of North Carolina
Johnston County

I, Angela Devlin, hereby depose and state as follows:

1. DNA evidence from the crime scene and from each suspect was analyzed using PCR
(Polymerase Chain Reaction).
2. PCR uses inanimate enzymes to replicate a section of DNA for analysis, making the process
easier to search for certain genes of interest. This will continue as long as the raw materials
that are needed to sustain the reaction are present. PCR can only replicate small sections of
DNA, when compared to DNA replication in nature.
3. Six basic components are needed for PCR: the DNA segment which will be copied, primers,
Taq polymerase, nucleotides, a chemical buffer environment, and a thermal cycler. The
thermal cycler is a machine which holds multiple tubes with multiple PCRs.
4. In the first step of PCR, the ingredients are mixed and the mixture is heated almost to boiling
point. This breaks the hydrogen bonds so the strands separate into single strands that can be
copied. This process of denaturing will take longer for longer strands, and vice versa.
5. The next step, annealing, involves primers (short, custom made strands of DNA) which are
made to bond to specific sites at the beginning and end of the segment being copied. If they
are not made correctly or the temperature is off, then primers may bind to random sites on the
DNA, which results in the wrong copy.
6. Extension, the last step of PCR, is when the polymerase copies the DNA segment quickly and
results in millions of copies which are made in minutes.
7. DNA evidence of 6 suspects was analyzed and compared to the victims DNA. There were
three different reactions. Each reaction used a different primer sequence. These are labeled as
1, 2, and 3. The PCR analysis matched the DNA profiles of the suspects to the evidence from
the crime scene (hair, blood on the lens, blood on the handle, and saliva on the handle).
8. Winnie Washedup was extremely jealous of her mother, as she was secretly in love with her
mothers boyfriend which could indicate motive for the murder.
9. According to results of the PCR analysis, the hair that was clenched in the victims hands
matched with Winnie Washedup, which makes her the likely killer.
Angela Devlin
April 18, 2017