Molecular Techniques Flashcards
Define the PCR
technique in which a small DNA sequence can be amplified allowing extremely small samples of DNA to be used for further testing
What are the tools needed for a pcr
Thermocycler Template of DNA Primer Taq polymerase dNTPS for DNA synthesis
What are the steps of the pcr
Denaturation at heat 94 to 98 to seperate DNA
Annealing at 50-68 to cool down sample allowing primers to to anneal to their complementary regions
Elongation at 72 to inc activity of taq polymerase
What are the applications of the pcr
Realtime pcr: basic type that amplifies but also addresses need for quantification of pcr product
Reverse transcriptase: reverse transcribes RNA in to DNA then amplified by pcr to detect RNA viruses
Define gel electrophoresis
Separation method based on differential of migration of charged particles in an applied electrical field
What are tools used in gel electrophoresis
Chamber Supporting material Ethidium bromide UV light source DNA ladder
What are the steps of gel electrophoresis
Agrose gel is poured horizontally in to electrophoresis chamber
DNA samples are pipetted in to sample wells
Application of an electric current at anodal side so DNA moves towards cathodal side
Smaller DNA fragments are at the bottom
DNA is visualized using ethidium bromide under uv light
DNA ruler is used for comparison
What are applications of gel electrophoresis
Analyze results of pcr
In blotting techniques
Illness in RFLP
Define blotting
Marking or staining and involve transfer of DNA,RNA or proteins on to a carrier in order to detect them
What are the tools needed for blotting
Sample containing material Tools for gel electrophoresis Blotting membranes(nitrocellulose) Probes( complementary to target and marked) Detection system
What are the steps of blotting
Electrophoretic separation
Blotting
Hybridization
Detection
What are applications of blotting
Southern: DNA for fingerprinting (STRs)
Northern: RNA to study mRNA
Western: proteins for antibody or antigen measurement
What are STRs
Certain sequences of non coding DNA and length varies from person to person
Define RFLP
Variation of DNA sequence can be recognized by restriction enzymes which cut DNA in to fragments of polymorphic lengths
What are tools used in RFLP
Dna extract
Restriction enzyme
Gel electrophoresis
What are restriction enzymes
Enzymes isolated from e coli and recognize double stranded DNA sequences and proceed to cut within yielding DNA restriction fragments
What are applications of RFLP
For analysis of mutations based on idea that mutations can create or eliminate restriction sites
Define dna sequencing
Technique that determines exact sequence of bases in a dna molecule using a dna sequencer
What are applications of DNA sequencing
Human genome project
Mutation testing
Define molecular cloning
Set of exp used to assemble recombinant DNA that can replicate in host organisms
What are the tools needed for Mol cloning
Dna from source to be cloned
Restriction Endonucleases
Cloning vectors
What are the steps of mol cloning
Restriction enzyme cuts DNA and vectors at restriction sites
Ligation of DNA fragments in to vector to form recombinant vector
Transfer in to cells
Grow host cells in culture
What are the applications of recombinant DNA
Protein production such as insulin replacement
Gene therapy:inducing a normal copy of the gene that is defective in to tissues that give rise to the pathology offering a potential cure for individuals with inherited diseases
