Biology: Biotechnology: PCR and Gel Electrophoresis
Amplifying DNA and analyzing fragment sizes
Biology: Biotechnology: PCR and Gel Electrophoresis
Amplifying DNA and analyzing fragment sizes
Biology - Grade 9-12
- 1
Explain the main purpose of PCR in biotechnology.
Think about what happens to the amount of target DNA during each cycle.
PCR is used to make many copies of a specific DNA region. This helps scientists analyze DNA even when the original sample is very small. - 2
A PCR cycle has three main temperature steps: denaturation, annealing, and extension. Describe what happens during each step.
During denaturation, the double-stranded DNA separates into single strands. During annealing, primers attach to matching sequences on the template DNA. During extension, DNA polymerase adds nucleotides to build new DNA strands. - 3
If PCR starts with 1 copy of a target DNA sequence and doubles perfectly each cycle, how many copies will there be after 6 cycles?
Use the pattern 1, 2, 4, 8, and continue doubling.
There will be 64 copies after 6 cycles because the number of copies is 2 to the 6th power, which equals 64. - 4
List four important ingredients in a PCR reaction and explain the role of each one.
Template DNA contains the sequence to be copied. Primers mark the start and end of the target region. DNA polymerase builds the new DNA strands. Free nucleotides provide the building blocks for the new DNA. - 5
Why is Taq polymerase commonly used in PCR instead of many other DNA polymerases?
PCR repeatedly heats the reaction close to the boiling point of water.
Taq polymerase is used because it can tolerate the high temperatures needed during PCR denaturation. Many other enzymes would lose their shape and stop working at those temperatures. - 6
A primer has the sequence 5'-ATG CCA TTA-3'. Write the complementary DNA sequence that would pair with it.
The complementary sequence is 3'-TAC GGT AAT-5' because A pairs with T and C pairs with G. - 7
Explain why primers must be specific to the DNA region being copied.
Primers act like starting points for DNA copying.
Primers must be specific so they attach only to the target DNA region. If primers bind to the wrong place, PCR may copy the wrong DNA fragment or produce extra unwanted products. - 8
What is the purpose of gel electrophoresis after PCR?
Gel electrophoresis separates PCR products by size so scientists can see whether the expected DNA fragment was made. It also helps compare DNA fragments from different samples. - 9
In gel electrophoresis, DNA fragments move toward the positive electrode. Explain why this happens.
Think about the charge on DNA and the charge of the electrode it moves toward.
DNA moves toward the positive electrode because DNA has a negatively charged phosphate backbone. Opposite charges attract, so the negatively charged DNA travels through the gel toward the positive end. - 10
A gel has three DNA fragments: 200 base pairs, 800 base pairs, and 1500 base pairs. Which fragment will travel farthest from the wells, and why?
The 200 base pair fragment will travel farthest because smaller DNA fragments move more easily through the gel matrix than larger fragments. - 11
A DNA ladder shows bands at 100 bp, 500 bp, 1000 bp, and 1500 bp. An unknown sample band lines up closest to the 1000 bp ladder band. Estimate the size of the unknown DNA fragment.
A DNA ladder is used like a size ruler for DNA fragments.
The unknown DNA fragment is approximately 1000 base pairs long because its band lines up closest to the 1000 bp marker in the DNA ladder. - 12
A forensic gel has these band patterns: crime scene sample has bands at 500 bp and 1200 bp, Suspect A has bands at 500 bp and 900 bp, Suspect B has bands at 500 bp and 1200 bp, and Suspect C has bands at 700 bp and 1200 bp. Which suspect matches the crime scene sample?
Suspect B matches the crime scene sample because both have bands at 500 bp and 1200 bp. - 13
A PCR experiment includes a positive control and a negative control. Explain what each control is used to check.
One control should work, and one control should show no amplification.
The positive control checks that the PCR reagents and conditions can produce the expected DNA product. The negative control checks for contamination because it should not produce a band. - 14
A student runs a PCR product on a gel and sees no band in the sample lane, but the positive control worked. Give two possible reasons the sample lane has no band.
Since the positive control worked, the overall PCR setup was probably functional.
The sample lane may have no band because the target DNA was not present in the sample or because the primers did not bind well to the sample DNA. Another possible reason is that the student forgot to add a needed PCR component to that sample tube. - 15
Compare the roles of PCR and gel electrophoresis in a DNA analysis workflow.
PCR makes many copies of a specific DNA region so there is enough DNA to study. Gel electrophoresis then separates the DNA fragments by size so the results can be viewed and compared.