DNA Manipulation (13.1) Flashcards
State what the PCR technique is used for
DNA amplification
Describe gel electrophoresis
Method used to separate and visualise nucleic acids and proteins according to their size
State what process DNA amplification uses to create a large quantity of DNA
PCR
State what PCR stands for
Polymerase Chain Reaction
State what DNA amplification enables
The rapid and accurate replication of target DNA
Describe ‘target DNA’
Particular region of a DNA molecule intended to be manipulated/studied
State what PCR is based on
The action of polymerases
Describe polymerases
Enzymes that catalyse the formation of polymers in nucleic acids
State the 2 different different groups of polymerases
- DNA polymerase
2. RNA polymerase
State the function of DNA polymerase
Acts to assemble DNA
State what polymerases can be classified as
Enzymes
Provide 2 examples of target DNA
- Specific gene
2. Microsatellite
Describe microsatellite
Short repeated sequence of nucleotides found at a defined locus on a chromosome.
State what makes microsatellites useful for DNA profiling
Variation in the number of repeats between individuals
State what the DNA polymerase enzyme uses as a template for building a new DNA strand
Each strand of the DNA double helix
State what each double stranded DNA copy is identical to
The original DNA strand
State what enzyme is involved in the unzipping of double helix of DNA
Helicase enzymes
Describe the function of primers
Determine the start and end points of a nucleotide sequence to be amplified
State why primers are so critical for the function of DNA polymerase
DNA polymerase can only attach to the end of a nucleotide chain that is base paired with the template strand
Describe taq polymerase
Thermostable DNA polymerase that is most commonly used in PCR
State from where taq polymerase was sought
Thermophilic bacteria
Describe the function of RNA polymerase
Acts to assemble RNA
State what RNA polymerase synthesises in cells
mRNA, rRNA and tRNA
State how RNA polymerase synthesises RNA strands in cells
Transcription of genes
State whether or not RNA polymerase requires a primer to anneal to the DNA template strand to start synthesis
No.
State until when RNA polymerase adds nucleotides
STOP codon is reached
State whether RNA polymerase or DNA polymerase functions faster
RNA polymerase works much more slowly
Describe reverse transcriptase
DNA polymerase that synthesises single-stranded DNA using single-stranded RNA as a template
State what the function of reverse transcriptase is the reverse of
The usual transcription process in which DNA is transcribed into RNA
State what reverse transcriptase is used to produce
DNA molecules that can be amplified by PCR for further analysis
State what reverse transcriptase is also used to make and from what
Complementary DNA (cDNA) from mRNA
State whether or not complementary DNA produced by reverse transcriptase includes or does not include introns
Introns are sliced out. Therefore, does not include.
State what PCR is a method of
Amplifying specific target sequences of DNA
State whether or not taq polymerase is used in the PCR method
Yes.
State why taq polymerase is used in the PCR method
Resists changes in temperature required during PCR
State what each cycle of PCR produces
2^n DNA
State what a PCR mixture requires
- DNA
- Free nucleotides
- Heat resistant DNA polymerase
- DNA primers (x2)
State whether or not target DNA to be amplified is also included in the PCR mixture
Yes.
State why free nucleotides are included in the PCR mixture
Used to build new DNA strands
State why heat-resistant DNA polymerase are included in the PCR mixture
Elongate the new DNA strands by adding the free nucleotides
Provide an example of a the heat-resistant DNA polymerase commonly used throughout PCR
Taq polymerase
State why DNA primers are complementary to the ends of the target DNA
Specify start and finish of the DNA sequence to be amplified
State why DNA primers are created
To form hydrogen bonds with complementary base pairs on either end of DNA sequence to be amplified
State where the PCR mixture is placed
DNA thermocycler
State what DNA thermocycler does for the PCR mixture
Alters the temperature over three steps
State the 3 major steps of PCR
- Denaturation
- Annealing
- Extension
Describe denaturation as the first step in the DNA thermocycler
Sample heated to 95C
State why the sample is first heated to 95C when placed in the DNA thermocylcer
To break hydrogen bonds between the two strands of double-stranded DNA to obtain single strands
Describe annealing as the second step in the DNA thermocycler
Temperature is reduced to 50-60C
State why the temperature of the DNA thermocycler is decreased to 50-60C in the second stage
Allows primers to bind to complementary sequences on opposite strands at end of target DNA sequence
Describe extension as the third step in the DNA thermocycler
Temperature is increased to 72C
State why the temperature of the DNA thermocycler is increased to 72C
Allows taq polymerase to attach to the primers on the DNA strands
State approximately how many times the heating and cooling cycle is repeated in the DNA thermocycler
Up to 50 times
State what the second stage of the DNA thermocycler process is known as
Annealing
State what the first stage of the DNA thermocycler process is known as
Denaturation
State what the third stage of the DNA thermocycler process is known as
Extension
State what gel electrophoresis can be used to do (in addition to separating DNA and RNA)
Separate proteins of different sizes
Describe when gel electrophoresis is commonly performed
After DNA amplification
State what gel electrophoresis allows scientists to do
Separate DNA fragments in a sample based on size
Describe gel electrophoresis
Technique used for separating fragments of nucleic acids
State why DNA is negatively charged
Negative charge on the phosphate group in each nucleotide
State what occurs to negatively charged DNA molecules when an electric current is applied to the electrophoresis gel
Negatively charged DNA molecules move towards positive terminal
State 3 reasons for which gel electrophoresis is used to compare DNA fragments
- DNA screening
- Confirming the correct gene has been amplified in PCR
- Identifying DNA fragments obtained by restriction enzyme digestion
State what gel involved in electrophoresis is composed of
Agarose
Describe agarose
Purified form of agar
State at what terminal of the chamber the wells are situated
Negative terminal
State where each DNA sample is loaded into
Within the gel of one of the wells
Describe DNA ladder
Set of DNA molecules of known size used to determine the size of other molecules
State what can be used in the gel electrophoresis process to estimate the length of the sample DNA fragments
DNA ladder
State what the gel placed in an electrophoresis bath is covered in
Controlled pH solution
State what the controlled pH solution introduced to the gel placed in an electrophoresis bath contains
Ions to conduct an electric current
State what the electrical current introduced to the gel electrophoresis bath causes
Negatively charged DNA fragments to migrate towards the positive terminal of the chamber
State what fragments move fastest in the gel when an electrical current is applied
Smaller fragments
State how DNA fragments are detected
Stain application that binds to DNA
State 2 examples of stain applied to detect DNA fragments
- Methyl blue
2. Fluorescent
State what the smearing of bands in the gel indicates
Overloading of wells/degradation of the DNA or protein
State the 3 ways PCR and electrophoresis can be used to detect a mutant allele
- Isolate individual’s DNA
- Use PCR primers complementary to DNA sequences
- Compare amplified DNA molecules
State why PCR primers that are complementary to the DNA sequences on either side of the site of the mutation are used to detect a mutant allele
Amplify DNA
Describe well
An indent in the gel into which a DNA sample is loaded
Describe standard ladder
A mixture of DNA fragments of known length that are used in order to infer the size of the fragments in the sample
Describe agarose gel
A sponge-like gel used in gel electrophoresis that contains pores for DNA fragments to move through
Describe buffer
An ion-filled solution that carries current though agarose gel
Describe electrode
Conductors of electricity that enable current to pass through gel
Describe band
A line seen in the gel after running gel electrophoresis that corresponds to a collection of DNA fragments of a specific size
Describe ethidium bromide
A fluorescent dye that binds to DNA fragments in a gel and allows them to be visualised
Describe recognition site
A specific target sequence of DNA upon which a restriction enzyme acts
