Biotechnology Lab Exam

What does PCR stand for, and why is it often called “molecular photocopying”?

Polymerase Chain Reaction, or PCR, is often referred to as “molecular photocopying” because it is a method used to quickly and easily copy segments of DNA.

What are the five main ingredients required for PCR?

• DNA Template to be copied

• Artificial made primers that bind to sections of DNA desired to be copied

• DNA nucleotide bases

• Taq polymerase

• Buffer solution

What is the role of the DNA template in PCR?

The template DNA is the original DNA the contains the target sequence. PCR copies the target region by using the template strands as the pattern for making new complementary strands

What is the function of primers in PCR?

Primers are short single stranded DNA sequences that bind (anneal) to the template strand on both sides of target region. Provide a free 3’ OH end so DNA polymerase can start adding nucleotides. They determine what DNA segment gets amplified.

Why are nucleotides added to the PCR reaction?

Are added because they are the raw materials DNA polymerase uses to build new DNA strands

What enzyme is responsible for synthesizing new DNA during PCR, and why is it used instead of other DNA polymerases?

Taq DNA Polymerase is used because it can withstand the high heat and not denature during the PCR process, unlike most DNA polymerase.

What machine is used to carry out PCR?

A thermal cycler: it rapidly changes temperature for denaturation, annealing, and extension.

What happens during the denaturing step of PCR?

The sample is heated. It causes the DNA to denature, or separate into two pieces of single-stranded DNA. The heat does this by breaking the hydrogen bonds that connect the two strands.

What happens during the annealing step of PCR?

The sample is cooled. When the temperature is lowered, DNA primers can attach to the template DNA.

What happens during the extending step of PCR?

Temperature is raises again. Taq polymerase synthesizes two new strands of DNA, using the original strands as templates. Nucleotides are added to the PCR reaction for Taq polymerase to use in synthesis. This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA

What type of DNA molecules are produced after one PCR cycle?

• 2 double stranded DNAs.

Why is PCR repeated multiple times?

Because each cycle roughly doubles the amount of target DNA. Repeating cycles 20-40 times, creating exponential amplification (2^n growth) more than one billion each copies of the original DNA segment

Approximately how many cycles of PCR are typically performed?

20 to 40 times

How many copies of DNA can PCR produce after many cycles?

More than one billion exact copies

How long does PCR usually take to complete?

Only a few hours to complete

How can the results of PCR be visualized?

• Via gel electrophoresis

  • Load PCR products into an agarose gel

  • Apply electricity

  • Stain DNA

  • View bands under UV/blue light

List three common applications of PCR.

Forensics, genetic testing, and diagnostic tests

How do restriction enzymes help bacteria defend against viral infections?

Bacteria use restriction enzymes to “cut up” their DNA where the viral DNA is located. Bacteria protect their own DNA from the restriction enzymes via methylation.

What is methylation, and how does it protect bacterial DNA?

Methylation is when methyl groups are added to sections of the DNA. Methyl groups mask restriction enzyme recognition sites (specific nucleotide sequences that are 4-8 nucleotides in length) on the DNA, thereby preventing the bacterial DNA from getting cut. Where the viral DNA is, however, there will be no methylation. This means that the restriction enzymes will be able to bind to recognition sites around the viral DNA and remove, or cleave the viral DNA.

What are restriction enzyme recognition sites?

Specific nucleotide sequences that are 4-8 nucleotides in length, where restriction enzymes bind and cut

What happens when a restriction enzyme binds to an unmethylated recognition site?

The enzymes cuts the DNA at/near that site, producing sticky ends (overhands) or blind ends (straight cut)

Why was the discovery of restriction enzymes important for molecular genetics?

It made cloning, DNA mapping, and sequencing possible. Scientists can genetically modify bacterial DNA using restriction enzymes and transformation, can manipulate bacterial DNA to add desired genes, producing a recombinant plasmid.

List three biotechnology applications of restriction enzymes.

Cloning, DNA mapping, and sequencing

Why are restriction enzymes useful for cloning and DNA mapping?

• Cloning: create compatible ends in plasmid and insert so DNA pieces can be joined together

• Mapping: cutting DNA with enzymes produces fragmen sizes that reveal locations of sites (a “map” of where recognition sequences occur)

Why is EcoRI considered an important tool in biotechnology research?

• High specificity, reliability, compatibility, commercial availability

• It recognizes a specific sequence G|AATTC

• Produces sticky ends, useful for cloning

• Became foundational in recombinant DNA techniques

What does it mean to genetically modify bacterial DNA?

• To change bacterial DNA by

  • Insertion a new gene

  • Removing a gene

  • Alternating and existing gene often done by introducing recombinant plasmids into bacteria

What is a recombinant plasmid?

A plasmid that contains DNA from another source inserted into it: plasmid + target gene

What role do restriction enzymes play in genetic modification?

• They cut both

  • Plasmid

  • Target DNA (gene of interest) creating compatible ends so the gene can be inserted into the plasmid

What is a recognition site?

A specific DNA sequences a restriction enzyme recognizes and binds to before cutting

What are “sticky ends”?

Refer to the cut DNA. ends are called “sticky” because they will want to form hydrogen bonds where the cuts where made; this is beneficial as it helps to hold the DNA together

Why do sticky ends “fit together like puzzle pieces”?

The target gene is insured at a specific restriction site that has been sliced by the same restriction enzymes. This will allow for complementary sticky ends and the gene will fit in like a puzzle piece

What happens when the target gene and plasmid are incubated together?

The recombinant plasmid can be incubated with bacteria so that the bacteria can uptake it through transformation

What enzyme permanently joins the target gene to the plasmid?

DNA ligase forms phosphodiester bonds in the sugar phosphate backbone, “sealing” the DNA

What is formed after ligase joins the target gene and plasmid?

A recombinant plasmid with the inserted target gene

What is transformation?

A cell or organism that acquires new DNA also acquires new characteristics and is said to be transformed.

What happens after bacteria take up the recombinant plasmid?

• They bacteria may 

  • Replicate the plasmid

  • Express the inserted gene if the plasmid has the right promoter to make the protein

  • Gain new traits like antibiotic resistance makers

Why is transformation important in biotechnology?

• It allows bacteria to

  • Make many copies of a gene (gene cloning)

  • Produce useful proteins (insulin, enzymes)

  • Test gene function in living cells

Why must the plasmid and target gene be cut with the same restriction enzyme?

Using the same enzyme creates matching ends (compatible ends or blunt ends), so the insert can fit into the plasmid for ligation

What would happen if different restriction enzymes were used?

• Often the ends would be incompatible

  • The insert would not anneal properly

    • Ligation would be inefficient or impossible unless the two enzymes create compatible ends, which is not always possible

Why is DNA ligase necessary if sticky ends already bind?

Sticky ends make temporary hydrogen bonds between bases. DNA ligase makes permanent covalent bonds in the backbone so the plasmid doesn’t fall apart.

What is the overall purpose of creating a recombinant plasmid?

• To carry a gene of interest into a host cell so the gene can be 

  • Copied (cloned/amplified)

  • Expressed (produce protein)

  • studies/manipulated

What happens to DNA when it is cut by a restriction enzyme?

The DNA is cleaved into fragments at specific recognition sites

What information does gel electrophoresis provide about PCR-amplified DNA fragments?

• Determine their relative sizes

• Whether amplification happened (bands present or not)

• Approximate fragment size

• purity/specificity ( one clear bands vs multiple bands/nonspecific products)

How could restriction maps be useful in cloning or genetic engineering?

• Choose enzymes that cut at useful locations

• Predict fragment sizes after digestion

Confirm an insert is present and in the right orientation (by expected band pattern)

Why does DNA move during gel electrophoresis?

DNA has a negative charge so it migrates towards the positive electrode

How does gel electrophoresis separate DNA fragments?

Gel electrophoresis can separate DNA fragments based on their size and charge; smaller DNA move more easily through gell pores while larger fragments more more slowly.

During gel electrophoresis, which size of DNA (long vs short fragment) will move the furthest? Why?

Short fragments move the furthest because they experiences the least amount of resistance

In which direction does DNA move during gel electrophoresis?

Moves towards positive electrode

What if you were performing gel electrophoresis and the molecule you were examining moved towards the negative electrode? What does this mean about the charge of the molecule you are analyzing?

It must be positively charged