Genetic Lab Techniques Flashcards
What are the three types of genetic lab techniques?
- Nucleic Acid manipulation; joining strands, separating strands, chopping into pieces
- Recombination and cloning
- Sequencing and function
What are some ways to denature dsDNA to separate strands?
Heat or immerse dsDNA in high concentration salt solution or high pH solution
Hydrogen bonds connecting two strands are disrupted and the strands separate
Melting temperature of DNA
Temperature required to separate DNA strands
DNA with more G-C bonds have higher Tm
95 deg C is generally sufficient to denature any DNA sequence
How does denatured DNA compare to dsDNA?Less vis
Denatured DNA is less viscous, denser, more able to absorb UV light than dsDNA
Nucleic Acid Hybridization
Techniques that use the spontaneous association of separate strands of nucleic acids to any complementary nucleotide sequence when not in a denaturing environment
DNA-DNA, DNA-RNA, RNA-RNA
Scientists can identify sequences by binding a known sequence with unknown sequence
Restriction enzymes
Digest (cut) nucleic acids only at certain nucleotide sequences along a chain (endonuclease)
These sites are called restriction sites or recognition sequences
- Typically a palindromic sequence 4-6 bp long
Usually uneven cleaving, leaving sticky ends for hybridization and then joined by DNA ligase
How can restriction enzymes be used in the lab?
Any two pieces of DNA fragments cleaved by the same restriction enzymes can be joined together, regardless of origin
Can create recombinant DNA (DNA library) for the purpose of DNA cloning
Gene cloning
Technique used to make many copies of DNA for sequencing, recombination, or manipulation
One copy of DNA is generally too small to be useful
Can be accomplished by:
1. Bacteria and a cDNA library
2. Polymerase chain reaction (PCR)
What is a clone library?
Bacteria grown with expression of certain recombinant DNA that has been inserted using a vector
How is antibiotic resistance used when introducing genes into bacteria?
If the vector that is used to introduce recombinant DNA contains antibiotic resistance, scientists can apply antibiotics to the bacteria to select for the bacteria that contain the vector
How is the lacZ gene used to determine which bacteria are expressing recombinant DNA?
The lacZ gene can be used in the original vector that the DNA fragment of interest is combined with. Some vectors won’t take up the DNA. Can use an endonuclease that inserts DNA fragment in the middle of lacZ gene, so if DNA fragment expressed, the lacZ gene doesn’t work and the bacteria don’t turn blue in presence of X-gal
How is hybridization used in creating a clone library?
Fluorescently or radioactively labeled complementary sequence of desired DNA fragment (probe) is used to label clones which have the gene. With radiolabeling, labeled clones expose radiographic film, and non-labeled ones do not
How can introns of Eukaryotic DNA be removed?
mRNA produced by organism (introns removed) can be reverse transcribed using reverse transcriptase
Produces complementary DNA (cDNA)
Add DNA polymerase to cDNA to produce ds DNA of fragment
How can a DNA library be created?
Take a DNA fragment and insert into bacterium using a vector. Reproduce and grow bacteria until a lot of clones are created.
Use antibiotic resistance, lacZ gene technique, and hybridization to further label and select for desired clones in library
Polymerase Chain Reaction (PCR)
DNA placed in mixture with 2 DNA primers for each strand, heat resistant polymerase, and nucleotides
- Denature DNA at 95 deg C (to single strands)
- Anneal to 60 deg C so DNA primers attach
- Heat to 72 deg C so polymerase copies DNA
- DNA amount has doubled!
- Repeat desired number of cycles to amplify
