Zoya Naz- Unit 5 Bacterial Transformation and Polymerase Chain Reaction

How did genetic engineering revolutionize medicine?

transformed medicine from reactive care into molecularly targeted, data-driven healing.

What is the donor plasmid?

A donor plasmid is a plasmid that donates the gene of interest for insertion into another plasmid or organism.

What are Restriction enzymes?

Restriction enzymes (also called restriction endonucleases) are molecular scissors—enzymes that cut DNA at specific, short nucleotide sequences.

What are DNA ligases?

DNA ligases are enzymes that act like molecular glue in genetic engineering—they join pieces of DNA together by forming covalent bonds between the sugar-phosphate backbones.

Where do foreign DNA come from?

Foreign DNA is any DNA that comes from a different organism than the one you’re working with. In genetic engineering, it is the gene or DNA segment you want to insert into a host.

What is a chimera?

In biology and genetic engineering, a chimera is an organism that contains cells or DNA from two or more different sources.

What does CaCl2 solution do to cells?

The CaCl₂ (calcium chloride) solution is commonly used in genetic engineering to make bacterial cells “competent”, meaning they are able to take up foreign DNA (like a plasmid) from their environment.

How does the host bacteria reproduce quickly?

The host bacteria used in genetic engineering (like E. coli) reproduce quickly because of their fast asexual reproduction process, called binary fission. This is a key reason why bacteria are used as hosts for cloning genes or producing proteins.

How can recombinant proteins be used?

Recombinant proteins are engineered proteins made by host cells that carry a foreign gene, used in medicine, research, and industry.

How did PCR revolutionize medicine?

PCR (Polymerase Chain Reaction) revolutionized medicine by allowing rapid, precise amplification of DNA

What are primers?

Primers are short, single-stranded pieces of DNA (usually 18–25 nucleotides long) that are essential starting points for DNA synthesis in processes like PCR (Polymerase Chain Reaction) or DNA replication.

What are dNTPs?

dNTPs are the building blocks of DNA, used in processes like DNA replication and PCR (Polymerase Chain Reaction). The term stands for deoxyribonucleotide triphosphates.

What is Taq DNA polymerase?

Taq DNA polymerase is a heat-stable enzyme that builds new DNA strands in PCR, allowing repeated cycles of DNA amplification.

What are the three steps of PGR?

Denaturation, Annealing, Extension (Elongation)

What temp does denaturation occur at?

94-95 degrees celcius

What happens during the denaturation phase?

• Heating the DNA

  • The reaction mixture is heated to 94–95°C.

• Breaking hydrogen bonds

  • The two strands of DNA are held together by hydrogen bonds between complementary bases.

  • High temperature breaks these bonds, without breaking the covalent backbone of DNA.

• Result

  • You get two single-stranded DNA molecules.

  • These single strands are now available for primers to bind during the next step (annealing).

What temp does annealing happen?

In PCR, the annealing step occurs at approximately 50–65°C, depending on the primers’ sequences and their melting temperatures (Tm).

What happens during annealing phase?

• Temperature is lowered

  • The reaction is cooled to 50–65°C (depending on the primers).

  • This lower temperature allows the primers to form stable hydrogen bonds with the single-stranded DNA.

• Primers bind to target sequences

  • Forward primer binds to one end of the target DNA on one strand.

  • Reverse primer binds to the other end on the complementary strand.

• Result

  • Each primer provides a starting point for DNA polymerase to begin synthesizing new DNA during the next step (extension).

What temp does elongation happen at?

In PCR, the elongation (extension) step occurs at approximately 72°C, which is the optimal temperature for Taq DNA polymerase.

What occurs during elongation stage?

• Optimal temperature

  • The reaction is held at ~72°C, which is the ideal temperature for Taq DNA polymerase to work efficiently.

• Primer-directed DNA synthesis

  • DNA polymerase attaches to the primer that has bound to the template DNA.

  • It adds complementary nucleotides (dNTPs) one by one, extending the DNA strand in the 5’ → 3’ direction.

• Result

  • A new DNA strand complementary to each template strand is formed.

  • After this step, the cycle is complete, and the DNA is ready for the next PCR cycle, doubling the amount of target DNA.

How does PGR multiply? (mathematical formula)

Mathematical formula for PCR amplification

If you start with N₀ DNA molecules and perform n PCR cycles, the total number of DNA molecules (N) is:

N=N0×2n\mathbf{N = N₀ \times 2^n}N=N0​×2n

Where:

• N0N₀N0​ = initial number of DNA molecules

• nnn = number of PCR cycles

• 2n2^n2n = doubling each cycle (assuming 100% efficiency)

How many cycles of PCR are typical?

In a typical PCR (Polymerase Chain Reaction) experiment, 20–40 cycles are usually performed.