Flashcards in Gene Regulation/Cloning Deck (23):
What type of analysis is performed with DNA templates?
Qualitative - you are exploring entire genes and sequences in between them
What type of analysis is performed with RNA templates?
Qualitative AND quantitative - exploring protein expression patterns
What type of DNA polymerase is used in labs for PCR?
What enzyme uses RNA as a template to make DNA? What is the new strand it produced called?
Reverse Transcriptase - new strand is called cDNA
What do restriction endonucleases do?
Cut ds DNA into smaller pieces, that are often palindromes; generates sticky ends and blunt ends
What does DNA ligase do?
Glues DNA strands together with a phosphodiester bond between adjacent nucleotides (requires ATP) - works much better with sticky ligation where hydrogen bridges can be formed
What are plasmids and how do they replicate?
Naturally occuring circles of dsDNA in bacteria that give them a special advtg (like antibiotic resistance)
They replicate independently from host chromosome, using a replicon
What are the two pathways bacteriophages can take after infecting a bacteria?
Lysogenic pathway - viral DNA incorporated into bacterial genome
Lytic pathway - viral DNA uses host's replication machinery to pack its DNA, then lyses host cells to release virions
What is the most common bacteriophage?
What are cosmids? Name two of them.
Hybrid of lambda phage and a plasmid - HUGE (inserts up to 45K bp)
BACs and YACs (bacterial/yeast artificial chromosomes) - contain huge DNA fragments
Describe basics of PCR (polymerase chain reaction).
Mix DNA template, all 4 nucleotides, 2 primers, and Taq-polymerase, then apply heating/cooling system (denaturatin, primer annealing, DNA extension, repeat)
Describe basics of Gel Electrophoreses.
Since DNA is negatively charged, will migrate to anode (+,red). In agarose or polyacrylamide gels, small fragments run faster than long runs (separation by size). Dye such as EtBr illuminates DNA.
How does reverse transcription-PCR work?
Mix RNA template, all 4 nucleotides, 1 primer, reverse transcriptase, apply heating/cooling system for primer annealing, cDNA synthesis, only 1 cycle!
Remove RNA with RNAse, then perform regular PCR to generate several copies of cDNA
Describe solid-phase DNA synthesis and why it is used.
Sequential addition of monomers to a growing chain that is linked to a solid support. Need an anhydrous environment, no template needed, used for short DNA fragments like primers or to design a desired sequence
What is Sanger Dideoxy DNA sequencing?
Mix DNA polymerase, template, and each nucleotide type (missing a 3' OH group) into 4 separate mixtures so that DNA synthesis terminates and all 4 tubes have DNA fragments of differing lengths
Can separate fragments by size to identify a DNA sequence
What is hybridization?
Identifying a sequence by annealing to it with a labeled probe (or antibody)
What are the three hybridization types?
Southern blot - DNA
Northern blot - RNA
Western blot - proteins
What is a microarray?
Fast screening for thousands of fragments in one sample - dots represent defined sequences
Black - neither sample is positive
Yellow - both samples are positive
Green - sample 1 is positive
Red - sample 2 is positive
What is transfection? What is it called if transfection is permanent?
Transfer of DNA into a host
What are host cells called when they are manipulated to better accept and keep foreign DNA?
How is most insulin in the USA produced?
Recombinant technology - pro-insulin human mRNAs are isolated, then RT-PCR is used to make a corresponding cDNA; Bacteria are transfected with a plasmid containing this cDNA sequence and forced to grow insulin that we harvest from them
What is a knockout animal?
Animal who has had DNA inserted into it that disrupts translation of functional proteins