A1. Because the gene’s promoter works in only one orientation, forcing RNA polymerase to bind and move in a single direction (5′→3′).
Q2. What is the template strand?
A2. The DNA strand is read by RNA polymerase to build complementary RNA.
Q3. What is the coding (sense) strand?
A3. The opposite DNA strand whose sequence matches the RNA (except U replaces T).
Q4. Can the “non-coding” strand of one gene act as the coding strand for another?
A4. Yes. Coding vs. template is gene-specific; different genes on the same DNA can use opposite strands.
Q5. What is pre-mRNA?
A5. The initial RNA transcript still contains introns and exons.
Q6. What is mature (processed) mRNA?
A6. Pre-mRNA after splicing removes introns, leaving only exons plus 5′ and 3′ UTRs.
Q7. What is the coding sequence (CDS)?
A7. The portion of mRNA that starts with AUG and ends with a stop codon; it specifies the protein.
Q8. Why does the NCBI “mRNA” FASTA show T instead of U?
A8. Because databases store a cDNA (DNA copy of mRNA) for convenience; in the cell, U is present.
Q9. What is cDNA?
A9. Complementary DNA made from mRNA by reverse transcriptase.
Q10. How is cDNA different from genomic DNA?
A10. cDNA contains only exons (no introns), reflecting mature mRNA.
Q11. Why is mRNA often called a “direct copy” of DNA?
A11. Its sequence matches the coding strand of DNA except U replaces T.
Q12. What does “complementary base pairing” mean?
A12. A pairs with T (or U in RNA), C pairs with G, allowing DNA replication and RNA transcription.
Q13. From which strand, leading or lagging is mRNA transcribed?
A13. From whichever strand contains the correctly oriented promoter; “leading/lagging” refers to DNA replication, not transcription.
Q14. What are flanking regions?
A14. Non-coding sequences upstream (5′) or downstream (3′) of the gene that regulate transcription.
Q15. What are transcript variants?
A15. Different mature mRNAs are produced from the same gene by alternative splicing or different start/stop sites.
Q16. Why can SNPs in introns or UTRs still affect disease risk?
A16. They can alter splicing, mRNA stability, or regulatory protein binding even if they don’t change the protein sequence.
DNA Direction, Strand Concepts & Primer Basics
Q1. What do 5′ and 3′ mean in DNA or RNA?
A1. They refer to the carbon numbers in the sugar backbone; the 5′ end has a free phosphate on carbon-5, the 3′ end has a free hydroxyl on carbon-3.
Q2. In which direction is DNA or RNA synthesized?
A2. Always 5′ → 3′, adding new nucleotides to the 3′ end.
Q3. Who decides which strand is the template for transcription?
A3. The promoter sequence determines RNA polymerase binding and direction, which fixes the template strand.
Q4. What is the difference between leading/lagging and sense/antisense strands?
A4. Leading/lagging describe DNA replication (continuous vs. Okazaki fragments).
Sense/antisense describe transcription (coding vs. template) and are gene-specific.
Q5. Why do we say upstream and downstream?
A5. They describe positions relative to the direction of RNA polymerase movement: upstream = toward the 5′ end (before the start site), downstream = toward the 3′ end (after the gene).
Q6. What is a forward primer?
A6. A short DNA oligo identical to the coding strand at the 5′ end of the target; it binds to the template strand and initiates synthesis in the natural 5′→3′ direction.
Q7. What is a reverse primer?
A7. A short DNA oligo complementary to the coding strand at the 3′ end of the target; it binds to the coding strand but is written in the 5′→3′ orientation so polymerase extends back toward the forward primer.
Q8. Why must primers be written 5′ → 3′ even when one binds the “other” strand?
A8. DNA polymerase can only extend from a 3′ hydroxyl, so every primer is synthesized and reported 5′→3′.
Q9. What GC content is generally preferred?
A9. Around 40–60 % for stable but not overly strong binding.
Q10. What is the typical melting temperature (Tm) range for PCR primers?
A10. About 55–65 °C, with forward and reverse primers ideally within ~2 °C of each other.
Q11. Why avoid long stretches of Gs or Cs at the 3′ end?
A11. They can form strong secondary structures or promote non-specific priming.
Q12. What are hairpins and primer–dimers?
A12. Hairpins are self-complementary loops; primer–dimers are forward/reverse primers annealing to each other—both reduce PCR efficiency.
Q13. How do 5′ and 3′ relate to primer design?
A13. Primers must match the orientation of DNA synthesis: forward binds near the 5′ end of the template, reverse binds near the 5′ end of the coding strand, but both are synthesized 5′→3′.
Q14. If a promoter is on the “bottom” strand, which primer is forward?
A14. Forward is still the primer that reads in the direction of transcription (matches the RNA sequence); “top” or “bottom” is arbitrary.
Some Basics of DNA Replication
Q1. What are the two strands of DNA called?
A1. The two strands are the plus (sense/coding) strand and the minus (template/antisense) strand.
Q2. What is the sense (coding) strand?
A2. It is the DNA strand whose sequence matches the mRNA (except it contains T instead of U).
Q3. What is the template strand?
A3. It is the opposite DNA strand that RNA polymerase reads to make the RNA.
Q4. Does RNA polymerase copy the sense strand?
A4. No. It copies the template (antisense) strand but produces an RNA sequence that matches the sense strand.
Q5. What does “strand +” mean on NCBI?
A5. It means the gene’s sense strand is the reference chromosome strand, so the sequence shown is already in the 5′ → 3′ coding direction.
Q6. What does “strand –” mean on NCBI?
A6. It means the gene’s sense strand is the opposite strand, so you must take the reverse complement of the displayed sequence to get the coding sequence.
Q7. What do chromosome coordinates like 11280841–11281330 represent?
A7. They represent the start and end positions of the gene on the reference chromosome.
Q8. If a gene is on the plus strand, how do you read its sequence?
A8. You read the given coordinates directly in the 5′ → 3′ direction to get the coding DNA.
Q9. If a gene is on the minus strand, how do you read its sequence?
A9. You must take the reverse complement of the chromosome sequence to obtain the coding strand.
Q10. Is the NCBI “gene sequence” the same as mRNA?
A10. For a plus-strand gene, it is the same as mRNA except DNA contains T instead of U and includes introns if you select the genomic sequence.
Q11. Why does mRNA look like the sense strand if RNA polymerase copies the template?
A11. Because base-pairing rules make the RNA complementary to the template and therefore identical to the sense strand (with U replacing T).
Q12. Is the sense strand always the leading strand?
A12. No. “Leading/lagging” refers to DNA replication, not gene orientation. A sense strand can be leading or lagging depending on replication origin.
Q13. Why is 5′ and 3′ orientation important?
A13. RNA synthesis and DNA replication both occur 5′ → 3′, so we always describe sequences in that direction for consistency.
NCBI vs UCSC Genome Browser
Q1. What is the NCBI Genome Data Viewer (GDV)?
A1. It is an interactive browser at NCBI that lets you explore gene locations, sequences, SNPs, and annotations directly from the NCBI RefSeq database.
Q2. What is the UCSC Genome Browser?
A2. It is a visualization tool from the University of California, Santa Cruz that displays a genome “track view” with many external datasets, conservation data, and custom tracks.
Q3. Are NCBI GDV and UCSC Genome Browser showing the same human genome?
A3. Yes, both can show the same genome assembly (e.g., GRCh38/hg38) but they use slightly different interfaces and track naming systems.
Q4. What is a “genome assembly”?
A4. A reference sequence of a species’ genome, such as GRCh38 for human, built from many sequencing projects.
Q5. Which browser directly uses RefSeq data?
A5. NCBI GDV primarily uses RefSeq, so the data matches NCBI’s reference gene records.
Q6. Which browser offers more external/custom tracks?
A6. UCSC Genome Browser offers broader custom track options and third-party data (ENCODE, conservation, etc.).
Q7. Which is easier for a simple gene or SNP look-up?
A7. NCBI GDV is often simpler for quick searches and official gene sequences.
Q8. Which is better for cross-species conservation?
A8. UCSC Genome Browser provides detailed conservation tracks (e.g., 100 vertebrate PhyloP/PhastCons).
Q9. What does “GRCh38/hg38” mean?
A9. GRCh38 is the Genome Reference Consortium Human build 38; “hg38” is UCSC’s name for the same assembly.
Q10. What does a chromosome coordinate like “chr16:11,280,841–11,281,330” mean?
A10. It specifies the start and end positions of a feature on chromosome 16 within the reference assembly.
Q11. What does “plus (+) strand” or “minus (–) strand” indicate in genome browsers?
A11. It shows which DNA strand contains the gene’s coding sequence relative to the reference assembly’s forward direction.
Q12. If a gene is on the minus strand, how is mRNA made?
A12. RNA polymerase uses the minus strand as the template to create an mRNA that matches the plus (coding) strand.
Q13. Do the coordinates run 5′→3′ along the reference genome?
A13. Yes, coordinates always increase along the reference genome’s plus strand.
Q14. Why might the PRM1 gene show an arrow pointing left in UCSC?
A14. Left arrows mean the gene is on the minus strand, so transcription proceeds opposite the reference coordinate direction.
Q15. What is the “Common dbSNP” track?
A15. It displays known single-nucleotide polymorphisms collected in NCBI’s dbSNP database.
Q16. What is the “OMIM Genes/Alleles” track?
A16. Links human genes and variants to clinical phenotypes from the OMIM database.
Q17. What is “MANE Select” in NCBI or UCSC?
A17. A single representative transcript agreed upon by NCBI RefSeq and Ensembl/GENCODE for consistent clinical reporting.
Q18. What is “ENCODE cCREs” in UCSC?
A18. Candidate cis-regulatory elements identified by the ENCODE project.
Q19. Which browser is preferred for official SNP IDs for publications or a PhD thesis?
A19. NCBI is often preferred because dbSNP IDs and RefSeq sequences are considered the primary reference.
Q20. Which browser is better for designing primers?
A20. Both can help, but NCBI’s Gene page links directly to sequence FASTA files and primer design tools.
Q21. Can you use both for a single project?
A21. Yes, many researchers cross-check a gene in NCBI for official sequence and in UCSC for extra regulatory and conservation information.
Q22. Are the three stacked gene rows in UCSC all the same gene?
A22. They can represent different annotations (RefSeq, GENCODE, MANE) but for the same genomic region.
Q23. What is a “track” in a genome browser?
A23. A visual layer of data (genes, SNPs, regulatory marks) that you can turn on or off.
Q24. Does NCBI show conservation tracks like UCSC?
A24. Yes, but UCSC provides more depth and custom alignments across many species.
Q25. For a PhD viva, which browser should you mention?
A25. Both are acceptable; ideally, explain that you used NCBI for official SNP/gene sequences and UCSC for regulatory or comparative data.
Comments
Post a Comment