PCR and gel elecrophoresis Flashcards
(29 cards)
what protein is stained purple in the gram stain?
- murein / peptidoglycan
in terms of membrane structure, what are the differences between gram positive and gram negative bacteria?
- gram positive have a thick peptidoglycan layer
- gram negative have a thin peptidoglycan layer and an out lipopolysaccharide membrane
what is ethanol’s role in the gram negative bacteria?
- removes outer lipopolysaccharide membrane on gram negative
- removed crystal violet / iodine complex from peptidoglycan layer
what is the last step in the gram stain and why is it important?
- counter staining with safranin to stain the gram negative bacteria pink
- safranin = pink colour!
what is the name of the complex that stains gram positive bacteria?
- crystal violet / iodine complex from
facts about DNA
- double stranded
- complementary
- specific to an individual
- base pairs
— (A-T) ~ 2 hydrogen bonds
— (C-G) ~ 3 hydrogen bonds
— A = adenine
— T = thymine
— C = cytosine
— G = guanine
— A + G are purines
— C + T + U are pyrimidine
what are restriction enzymes?
- protein isolated from bacteria that cleaves DNA sequences at sequence-specific sites
- this produces DNA fragments with a known sequence at each end
what do restriction enzymes do?
- cut the DNA double stranded at specific sequences
- wherever the DNA had this sequence, the restriction enzymes breaks it, leaving exposed based pairs of a known type
- they are used to chop DNA up ready for PCR or gel electrophoresis
- has sticky ends to easy attachment
what is PCR?
- polymerase chain reaction (PCR)
- a means of selectively amplifying a particular segment of DNA
- the segment may represent a small part of a large and complex mixture of DNA, e.g. a specific human gene
- can be thought of as of as a molecular photocopier
what do you need in order to do PCR?
- starting material (template strand, original sample)
- primers (25bp long, attach to the template)
- nucleotides (extending the strand after the primer)
- taq polymerase (bacterial polymerase, thermally stable, will not denature at a high temperature)
- MgCl2 / salt buffer (neutral salt, balanced)
what are the 3 stages of PCR?
- denaturation - 94 degrees, 30 seconds
- annealing - 60 degrees, 30 seconds
- elongation - 72 degrees, 60 seconds
what is step 1 of PCR?
denaturation of the template into single strands (heated to 95 degrees)
- DNA template and primers melt by disrupting the hydrogen bonds between complementary bases
- gives single strands of DNA
what is step 2 of PCR?
annealing of primers to each original strand (cooled to approximately 55 degrees)
- allows annealing of primers to the single strand of DNA template
- the polymerase bonds to the primer-template and begins DNA synthesis
what is step 3 of PCR>
extension of the new DNA (heated to 70 degrees)
- done after the final PCR cycle to ensure that any remaining single-stranded DNA is fully extended
- a thermally stable DNA polymerase (taq) adds complimentary nucleotides (extension) by forming bonds in the sugar-phosphate backbone (phosphodiester bonds)
what happens after you’ve completes all of these steps?
- repeat the steps agin
- after up to 40 cycles, over a billion copies of the target sequence can be produces from just one piece of DNA
what are the uses of PCR?
- allows a quantity of DNA to be amplified for analysis
- used to amplify small sections of DNA rapidly
- amplify DNA by using a primer (single stranded DNA typically 6-25bp in length)
- this is complimentary to the start of the sequence
why is PCR useful?
- developed in 1983
- common use across medical and forensic science
— parentage testing
— DNA fingerprinting
— diseased screening
— species identification
what are the steps of DNA profiling?
- obtain DNA sample
- create fragments of DNA using restriction enzymes
- amplify DNA - polymerase chain reaction (PCR)
- separate the fragments - gel electrophoresis
- visualise the fragments - stain them and compare them to a DNA ladder
what is an electrophoresis needed for?
- to use a UV light to see the results and photographed
gel electrophoresis
- DNA has a negative charge and is attracted to positive electrical current
- putting the sample in a gel that DNA can pass through then add the electrical current and buffer, which carries the charge
- DNA is sorted by size - smaller travel further faster, larger travel less and slower
- this process separates DNA segments according to their size
- big ones travel more slowly through the gel than smaller ones
DNA sequencing
- if a sample needs sequencing the fluorescently labelled bases are added to the PCR
- the PCR product is lined up and a laser shot through it to read the different coloured bases
- this gives the order of the DNA and compared to a known sequence to detect mutations or diseases causing genes
what is gel electrophoresis used to separate?
- different lengths of DNA
what do restriction enzymes do?
- cut DNA
- specific coded regions
what 2 roles does ethanol play in gram staining?
- removed the outer membrane on gram negative
- removes the crystal violet / iodine complex from peptidoglycan
