21.1 Producing DNA Fragments Flashcards
What is recombinant DNA technology and its significance?
Recombinant DNA technology involves manipulating, altering, and transferring genes between organisms. It enables:
- Better understanding of how organisms function.
- Designing new industrial processes and medical applications.
- Treating diseases by producing pure proteins, e.g., insulin, through genetically modified microorganisms.
What were the problems with extracting proteins from donors for medical treatment?
Issues included:
- Immune system rejection by the patient.
- Risk of infection from donor material.
- High costs of extraction.
What are the benefits of using recombinant DNA technology to produce proteins?
- Produces large quantities of pure proteins.
- Reduces the risk of rejection and infection.
- Utilizes microorganisms as factories to manufacture desired proteins.
What is recombinant DNA and a transgenic organism?
- Recombinant DNA: DNA from two different organisms combined together.
- Transgenic organism: An organism containing recombinant DNA.
Why can DNA from one organism function in another?
- Genetic code is universal: The same codons specify the same amino acids in all organisms.
- Protein synthesis mechanisms (transcription and translation) are essentially the same across organisms.
What are the stages involved in producing a protein using recombinant DNA technology?
Isolation of the DNA fragment containing the target gene.
Insertion of the DNA fragment into a vector.
Transformation: Transferring the DNA into host cells.
Identification of host cells that successfully took up the gene.
Growth/cloning of the host cells
What are the three methods to isolate a target gene?
- Using reverse transcriptase to convert mRNA into cDNA.
- Using restriction endonucleases to cut the DNA at specific sequences.
- Using a gene machine to synthesize DNA fragments from scratch.
How is reverse transcriptase used to isolate a gene?
- Identify cells producing the target protein (e.g., insulin-producing beta cells in the pancreas).
- Extract mRNA from these cells (mRNA is complementary to the target gene).
- Use reverse transcriptase to synthesize complementary DNA (cDNA) from mRNA.
- Use DNA polymerase to build the complementary strand of the cDNA, forming a double-stranded DNA molecule (the gene).
What is reverse transcriptase and its role in DNA isolation?
- Enzyme isolated from retroviruses (e.g., HIV).
- Catalyzes the production of DNA from RNA (reverse of normal transcription).
- Used to create complementary DNA (cDNA) from mRNA.
What are restriction endonucleases and how do they isolate genes?
- Enzymes from bacteria that cut DNA at specific recognition sequences.
- Recognition sequences can result in:
1) Blunt ends: Straight cuts.
2) Sticky ends: Staggered cuts with unpaired bases. - Sticky ends allow fragments to join together during cloning.
How does a gene machine create genes?
1) Determine the target protein’s amino acid sequence.
2) Derive the nucleotide sequence of the gene.
3) Feed the sequence into a computer.
4) The computer designs oligonucleotides (short DNA strands).
5) Oligonucleotides are synthesized, assembled into the full gene, and checked for errors
What are the advantages of using a gene machine to synthesize DNA?
- Can create any nucleotide sequence with high accuracy.
- Produces genes free of introns and non-coding DNA (efficient for prokaryotic cells).
- Fast production (as little as 10 days).
How is the synthesized gene prepared for use?
- Double-stranded gene is replicated using PCR.
- Sticky ends are added to facilitate insertion into a vector.
- Gene is stored, cloned, or transferred to organisms
What are the key features of restriction endonucleases?
- Recognise specific palindromic sequences in DNA.
- Cut DNA at recognition sites, creating sticky or blunt ends.
- Derived from bacteria as a defense mechanism against viruses.
