Recombinant DNA

Question 1

How do restriction enzymes recognize and cut DNA?

Answer 1

They recognize specific palindromic sequences in DNA and cut at or near these sites, creating either blunt ends or sticky ends.

Question 2

What are the steps to create a recombinant DNA plasmid?

Answer 2

1. Cut plasmid and gene of interest with the same restriction enzyme
2. Mix and allow sticky ends to anneal
3. Use DNA ligase to seal the backbone
4. Transform plasmid into host cells

Question 3

How can you identify restriction enzyme sites in a DNA sequence?

Answer 3

Look for the specific recognition sequence (often palindromic) of the enzyme (e.g., EcoRI: GAATTC) in the DNA strand.

Question 4

How do you determine if two restriction enzymes create compatible sticky ends?

Answer 4

Compare their cutting patterns and overhangs—sticky ends must have complementary single-stranded sequences to ligate.

Question 5

How do you determine the sizes of restriction fragments?

Answer 5

For linear DNA: Count how many times it is cut and measure each resulting fragment.

For circular DNA: One cut = linearized plasmid; two or more cuts = multiple fragments based on distances between sites.

Question 6

What is the difference between cDNA and genomic DNA?

Answer 6

cDNA is reverse-transcribed from mRNA → contains only exons (no introns).

Genomic DNA includes introns, exons, promoters, and all non-coding regions.

Question 7

How do you transcribe and translate a cDNA sequence into an amino acid sequence?

Answer 7

1. Transcribe the DNA to mRNA (replace T with U).
2. Use the mRNA codons to find corresponding amino acids using the genetic code.
3. Start translation at AUG (start codon) and continue until a stop codon is reached.