Exam 6 Flashcards
DNA Sequencing (Sanger method)
Don’t play every tune fast, silly
- DNA Denaturation
- Primer Annealing:
A primer, a short sequence of DNA complementary to one end of the target DNA, is allowed to bind to the single-stranded DNA. - Chain Extension:
DNA polymerase, an enzyme that synthesizes new DNA, extends the primer, adding nucleotides to the 3’ end. - Chain Termination:
The reaction mixture contains both normal dNTPs (deoxyribonucleotide triphosphates) and ddNTPs, which lack the 3’ hydroxyl group necessary for further nucleotide addition. When a ddNTP is incorporated, the chain growth is terminated. - Fragment Separation:
The resulting DNA fragments, each terminated by a ddNTP, are separated by size using gel electrophoresis. - Sequence Reading:
The order of the ddNTPs, which are often fluorescently labeled, is read to determine the DNA sequence.
Bioinformatics
Uses computer-based approaches to organize, share, and analyze data related to:
Gene structure
Gene sequence and expression
Protein structure and function
Whole-genome sequencing (shotgun cloning)
Genomic DNA is cut into fragments and contigs made
Entire chromosome is assembled by computer program
Fragments are aligned based on identical DNA sequences
BLAST (Basic Local Alignment Search)
Software application used to compare a segment of genomic DNA to sequences throughout major databases
Identifies portions that align with or are the same as existing sequences (similarity score)
“E Value”: Expect value
Based on number of matching sequences in database expected by chance
Plasmid
plasmids are small, circular DNA molecules used as vectors to carry foreign genes into cells, enabling scientists to manipulate and study gene expression.
They are essential tools for cloning, amplifying DNA, producing recombinant proteins, and gene therapy applications.
origin of replication
how gene of interest will get replicated to daughter cells
Selectable markers
genes that are proteins for the organisms required to survive in different environments, such as an ampicillin resistant gene that helps the bacteria we want in cows to survive antibiotics to kill the incorrect bacteria
Restriction enzymes
Produced by bacteria as a defense mechanism against bacteriophage
DNA-cutting enzymes
Bind to DNA at specific recognition sequence (restriction site) and cleaves DNA to produce restriction fragments
Enzyme cleaves both strands of DNA (digestion)
Palindrome
Symmetry exhibited by recognition sequences
Nucleotide sequence reads same on both strands
Restriction enzymes cut DNA in characteristic cleavage pattern
2 types of cuts
Sticky ends (cohesive ends):
Fragments produced with overhangs
Blunt ends:
Fragments produced with double- stranded ends
DNA Ligase
DNA fragments will seal phosphodiester backbone
Joins restriction fragments covalently to produce intact DNA molecules
Vectors: Carrier DNA molecules
Can replicate cloned D N A fragments in host cell
Must be able to replicate independently
Have several restriction enzyme sites to allow insertion of DNA fragment
Carry selectable gene marker to distinguish host cells that have taken them up from those that have not
Plasmid
Extrachromosomal double-stranded DNA molecule
Replicates independently from chromosomes within bacterial cells
Transformation
Plasmids are introduced into bacteria via transformation
2 main techniques:
Using calcium ions and brief heat shock to pulse DNA into cells
Electroporation: A brief but high-intensity pulse of electricity to move DNA into bacterial cells
Where does our gene of interest come from?
8 steps
Our gene of interest already exists in one bacteria species, so we take it from them and replicate it with PCR
- Gotta have polymerase
- Primers for specificity for our gene of interest
- Free nucleotides (dNTPs)
- Break hydrogen bonds at 95 degrees c
- Anneal the two separate strands of DNA for primer to bind to it
- Slightly increase temperature for polymerase to bind to strands
- New strands are formed!
- Around 30 cycles occur in one PCR
Dideoxynucleotides (ddNTPs)
ddNTPs are structurally modified versions of dNTPs for PCR
deoxyribonucleoside triphosphates, the natural building blocks of DNA.
Advantages of whole-genome sequencing
Provides a high-resolution, base-by-base view of the genome
Captures both large and small variants that might be missed with targeted approaches
Identifies potential causative variants for further follow-up studies of gene expression and regulation mechanisms
Delivers large volumes of data in a short amount of time to support assembly of novel genomes
Key Clue Used to Assemble Contigs
Overlap-Layout-Consensus (OLC) (used more in long-read assembly)
It finds overlaps between reads—where the end of one read matches the beginning of another.
pMMO
particulate methane monooxygenase
an enzyme in methylococcus capsulatus that catalyzes a reaction of methane and oxygen into methanol
3 polypeptides:
pmoC
pmoA
pmoB
blaC
the selectable marker in the recombinant E. coli with the pMMO gene
B-lactamase
ampicillin resistance
pmoCAB operon
no. of promoters
no. of mrna strands
no. of ribosome sites
no. of start/stop
no. of promoters 1
no. of mrna strands 1
no. of ribosome sites 3
no. of start/stop 3
