Exam 4: PCR and molecular techniques Flashcards
Describe the necessary reagents needed to execute a polymerase chain reaction (PCR).
-DNA template
-2 DNA primers
-DNA polymerase (Taq)
-Thermocycler
-A buffer to put the DNA in
-dNTP building blocks
Relate PCR primer design to the PCR product.
Primer design will orient a 5’ and 3’ end.
The primary goal of PCR primer design is to ensure the specific amplification of the target DNA sequence. Primers should be designed to anneal only to the intended target region, minimizing the likelihood of nonspecific amplification. This specificity is crucial to obtaining accurate and reliable PCR products.
Compare and contrast PCR with DNA replication in vivo.
PCR:
-Utilizes a heat-stable DNA polymerase (such as Taq polymerase) -The process typically involves denaturation, primer annealing, and extension steps.
DNA Replication in vivo:
-Involves a family of DNA polymerases and various other enzymes and proteins
-DNA replication in vivo is a more complex process, including multiple steps like initiation, elongation, and termination
Recognize the importance of short tandem repeats in the process of DNA fingerprinting
STRs exhibit a high degree of polymorphism, meaning that the number of repeats in the DNA sequence varies significantly among individuals. This polymorphism provides a unique genetic profile for each person, making STRs ideal markers for distinguishing between individuals.
Describe the role of restriction enzyme digestion when inserting a DNA fragment into a plasmid.
Restriction digest allows for insertion
of your DNA fragment into a vector.
Incubate the plasmid vector with the chosen restriction enzymes. These enzymes will cleave the vector at specific sites, generating linearized vector fragments. Incubate the DNA fragment with the same restriction enzymes used for vector digestion. The enzymes will cleave the fragment at corresponding recognition sites. The ligase catalyzes the formation of phosphodiester bonds between the complementary overhangs, resulting in the formation of a circular recombinant plasmid.
Compare and contrast Sanger sequencing with typical PCR.
-Sanger sequencing is used for determining the sequence of DNA.
PCR is used for amplifying specific DNA sequences.
-PCR sequencing uses a forward and reverse primer. Sanger sequencing uses one primer instead of two. The amplification process copies one strand but not the reverse strand.
-Both involve DNA amplification, DNA polymerase, and both use primers.
Define the role of dideoxynucleotides in a Sanger sequencing reaction.
DdNTPS lack a 3’ OH group which prevents further DNA chain elongation after incorporation. In a Sanger sequencing reaction, a mixture of regular dNTPs and a small amount of ddNTPs are used. This allows for the synthesis of DNA strands of varying lengths. As a result, a set of DNA fragments is generated, each terminating at a specific nucleotide position.
By generating fragments of varying lengths, the sequencing reaction provides information about the positions of specific nucleotides in the DNA template.
What are the three steps of thermocycling?
-Denaturation: heats the reaction to 94-98 C for 20-30 seconds. It causes DNA melting of the dsDNA template into ssDNA molecules.
-Annealing step: temperature is lowered to 50-65 C for 20-40 seconds allowing annealing of the primers to the ssDNA template.
-Extension/elongation step: Taq polymerase has its optimum activity temperature around 72 C. The extension time depends on both the DNA polymerase used and the length of the DNA fragment to be amplified.
Gel Electrophoresis
-Molecules are separated based on size and electrical charge
-Small molecules run quickly, so they are farthest.
-If you run an electrical current through your gel, all your negatively charged molecules (such as DNA) will be attracted to the positive end.
1) DNA samples of different sizes are placed in wells in agarose gel.
2) An electrical current is passed through the gel.
3) A dye specific for nucleic acids is added to the gel.
4) DNA fragments appear orange under UV light.
Southern Blotting
-Transfers banding/smear onto membrane.
-Membrane is hybridized with a radio labeled probe which is complementary to your fragment of interest.
-Membrane with bound probe is exposed to film.
List the steps of a polymerase chain reaction
1) A DNA molecule with a target sequence to be copied is heated to 90 C to denature it.
2) When the mixture cools, artificially synthesized primers bond to the ssDNA.
3) Heat-resistant DNA polymerase uses dNTPs to synthesize two new strands of DNA.
4) The process is repeated, doubling the amount of DNA.
5) By repeating the process, many copies of the DNA can be produced in a short time
DNA fingerprinting
DNA fingerprinting is a laboratory technique used to determine the probable identity of a person based on the nucleotide sequences of certain regions of human DNA that are unique to individuals.
What are some problems with restriction sites?
-Short (~6 bp) sequences will occur randomly throughout the genome in high frequency. This will limit the size and type of genes you can introduce.
-Incompatibility of sites
What are the two characteristics that separate molecules during gel electrophoresis?
Size and charge :)
