6.1.3 Manipulating Genomes Flashcards
What is a genome and what does it consist of?
-all of the genetic material within an organism
-made up of DNA in the nucleus contained within chromosomes and mitochondria
Explain what short tandem repeats are
-exist within introns, telomeres and centromeres
-short sequences of DNA that are repeated many times–} known as microsatellites made up of repeated regions of 2-4 bases
-always appear on the same position on the chromosomes but the number of repeats vary per individual–} different length repeats inherited from each parent= unique pattern
-more closely related you are, the more likely you are to have similar patterns i.e identical twins(allows for family mapping/paternity testing)
Why do STRs exist in introns and not exons?
-because our exons are highly conserved as they are essential for protein synthesis, whereas intron are not expressed as they are non-coding regions of DNA
What is a DNA profile?
-a unique DNA sequence that allows an individual to be identified based on the STRs at each locus on their chromosomes
What are the steps of making a DNA profile?
-DNA extraction
-Amplify DNA using PCR
-Cut up DNA fragments using restriction enzymes
-Separate DNA using gel electrophoresis
-Visualisation through hybridisation
-Comparison of DNA samples or sequencing
DNA extraction
-break up the sample using a pestle and mortar to break up the cell wall
-make up a solution of detergent(breaks up cell membrane and nuclear envelope), salt(clumps up the DNA) and distilled water and add the cells to the solution beaker
-incubate the beaker at 60 degrees Celsius (denatures digestive enzymes)
-put beaker into an ice bath to cool the mixture down
-filter the mixture to isolate DNA extract
-add protease to break down histones and expose the DNA
-slowly dribble cold ethanol down the side of the test tube(causes DNA to precipitate as it is insoluble in alcohol)–} can be extracted with a glass rod
Define all the substances needed for PCR
-Taq DNA polymerase= enzyme found in bacteria from thermal hot springs–} can withstand high temps and doesn’t denature at 95°(many cycles of PCR can be carried out without needing new enzymes each time), used to create new DNA strands
-Primers= short, single-stranded pieces of DNA complimentary to the start of the sequence to be amplified
Describe how DNA is amplified using PCR
-PCR is used to create many copies of DNA from a fragment
- 95°C:
-DNA mixture is heated to 95° for 30 seconds–} breaks the hydrogen bonds and separates the 2 strands of DNA
- 55°C:
-DNA mixture is cooled
-complimentary primers anneal to the end of each strand
- 72°C:
-DNA mixture is heated for optimum conditions for Taq to speed up the reaction
-Taq DNA polymerase lines up free nucleotides onto the primer complimentary the template strand
What are the results of one PCR cycle?
-2 new double stranded fragments of DNA identical to the original sample
(the amount of DNA doubles each cycle i.e 16 cycles= 2 to the power of 16)
Explain what restriction enzymes are?
-some sections of DNA have palindromic sequences of nucleotides(consist of antiparallel base pairs)
-restriction enzymes will recognise and cut the DNA at the restriction site
-the active site of a restriction enzyme is specific to the complimentary shape of the restriction sites
How do restriction enzymes work?
-the DNA sample is incubated with the specific restriction enzyme
-hydrolysis reaction occurs
breaking sugar phosphate backbone of DNA via breaking down phosphodiester bonds using water
-reaction often results in a ‘staggered cut’ leaving exposed bases at the end of each fragment called sticky ends –} can be used to bind the DNA fragment to any other DNA fragment that has complimentary sequence at the sticky ends
Separating DNA using gel electrophoresis
-uses an electrical current to separate out DNA fragments
-gel tray is added to gel box/tank, the end with wells is positioned next to the cathode(negative)
-gel tray is immersed in a buffer solution(keeps reaction alkaline which maintains the pH and helps carry the current)
-a set vol of loading dye is mixed with each sample to be separated(ensures DNA sink to the bottom of the wells and helps visualise the DNA)
-ladder(DNA sample of known fragment lengths) is loaded into the first well as a control
-gel box is closed and connected to power supply(cause current to run through gel)
-negative DNA fragments move through mesh like structure of the gel–} rate of movement depends on mass/length of DNA fragment, shorter=quicker and vice versa
How can electrophoresis be carried out on RNA fragments and proteins?
-follow the same basic DNA method for RNA
-proteins can be negatively OR positively charged, so they are mixed with a chemical(SDS) to coat the protein with a negative charge and denature it so it is at its primary structure
What is southern blotting?
-after electrophoresis, fragments are placed in an alkaline buffer solution which denatures the DNA so it is single-stranded(exposed DNA stands so probes can bind via CBP)
-through southern blotting, strands are transferred to a nylon membrane in exactly the same position relative to the gel–} fixed in place using UV light or heated at 80°C
Visualisation through hybridisation
-probes are short DNA/RNA fragments complimentary to known STRs
-radioactive or fluorescent DNA probes are now added to the DNA fragments on the membrane
-bind to complimentary strands of DNA and identify STRs
