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
Quantitative PCR (qPCR)
AKA real-time PCR
PCR used to quantify the amount of DNA in each cycle using a fluorescent dye or probe that binds to DNA and measures its concentration in each cycle
Sanger Method for DNA sequencing
AKA Chain termination method
DNA repeatedly replicated in vitro, separated into four tubes containing nucleotides, primers, enzymes, and a different ddNTP which terminates the replication for each type of base
Each of 4 tubes added to different lane in gel electrophoresis and sequence can be read by bands
“Next Generation” DNA Sequencing Example
Fluorescent nucleotides used with different colors for each base to perform DNA replication in vitro. Computerized reader can convert color-coded DNA molecule to sequence.
DNA Microarray or Gene Chip
Compares levels of mRNA between two cells and/or conditions
Microarray contains probes for hundreds or thousands of genes
One mRNA labeled red and other mRNA labeled green- then both sets incubated with same gene chip. Visualize results: Downregulated- red, upregulated- green, same expression- yellow
Probe for DNA microarray
Small DNA Sequences complimentary to mRNA
What are ways scientists can determine gene function?
- Observe gene conservation or evolution among different species
- Direct manipulation of genes to observe results of a reduction, elimination, addition of gene expression
Genetic Knockout
Can refer to a cell line or animal in which a target gene is deleted from the genome
Phenotype can be analyzed for changes, providing information about gene function
What cells are modified to create a knockout animal?
Need to modify and knock out the genes from gametes or from embryonic stem cells and grow the animal from a zygote
How can RNA Interference be used to reduce gene expression?
RNAi is process in which small pieces of RNA bind to mRNA molecules to prevent translation or mark for degradation
RNAi can be used on certain genes to prevent gene expression by preventing translation
How can gene expression be amplified?By modifyin
By increasing the strength of the promoter before a gene, or decreasing strength of a repressor
Restriction fragment length polymorphism (RFLP) Analysis
Can be used to uniquely identify individual’s by comparing DNA samples (DNA fingerprint)
Each individual has distinct restriction sites (human pop. Polymorphic for restriction sites)
Fragment DNA with endonucleases and perform Southern Blot to get unique band pattern for individual
Single nucleotide polymorphism (SNPs)
DNA sequence variation occurring when a single nucleotide in genome differs between members of a species
Can be a DNA fingerprint for individual’s genome
Gene Therapy
Genetic manipulation of an affected individual’s DNA to replace defective allele of gene with wild type, or correctly functioning allele
Highly experimental, has had some dangerous side effects
Genetic Screening
Patients can be screened for genetic disorders to see if they are carriers or could pass on to offspring
Bacteria can be genetically screened for antibiotic resistance- faster, cheaper, and more accurate than traditional culture growing
Biometry
Use of statistical methods to understand biological data
E.g. population genetics to correlate genes with medical conditions, better understand global health and epidemiology
Ethical implications of DNA technology
Ethical altering of DNA of agriculture, plants, other animals, and humans
“Designer” babies: parents choosing the sex and physical or mental attributes of offspring
Modifications of embryonic stem cells- controversy of “playing God” and origins of life
Where are prokaryotic proteins produced?
Always in the cytosol because they do not have membrane-bound organelles or endoplasmic reticulums
What is a disadvantage of developing a gene knockout model?
Cannot be used to study genes that are necessary for embryonic development
Where does the phosphodiester bond of DNA and RNA connect to on the nucleotides?
Attaches to phosphate group which is attached at the 4th Carbon on the pentose backbone of one nucleotide and to the hydroxyl group on the 3rd Carbon on the pentose backbone of the next nucleotide.
How does the structure of a ddNTP differ so that phosphodiester bonds cannot be created as they normally are?
Has no hydroxyl on the 3rd Carbon of the pentose sugar
RNA has no hydroxyl on the 2nd Carbon, but this is not the location of the attachment to the phosphate of the previous nucleotide in the phosphodiester bond
