2. Studying DNA Flashcards
What at first was thought to be the carrier of genetic material and why?
Protein
Had greater diversity
Griffiths Experiment
Smooth (bad) and rough (not bad) bacteria
Found that harmless R bacteria would be transformed by taking up genetic material from S and became harmful
What are the bases in DNA?
Guanine
Adenine
Thymine
Cytosine
What are the bases in RNA
Guanine
Adenine
Uracil
Cytosine
What are the 3 main constituents of necleotides that make up DNA?
Base
Sugar (deoxyribose)
Phosphate
What is Chargaff’s rule?
%A = %T %G = %C
What are the advantages of DNA over RNA?
DNA is more stable
What are the key characteristics of DNA?
Capacity for replication
Capacity to store information
Capacity to express information
Capacity for variation by mutation
What is the structural difference between RNA and DNA’s sugar?
Ribose has an O molecule on the bottom of the 2’C
Deoxyribose doesn’t
What bond holds the DNA backbone together?
3’-5’ phosphodiester bonds
Which of the bases are purines?
Adenine
Guanine
Which of the bases are pyrimidines?
Uracil
Thymine
Cytosine
Why are H bonds good for joining strands of DNA?
Week enough to be seperated in localised areas without using too much energy
Strong enough overall to hold it together
What’s at the 5’ end of DNA?
A phosphate group
What’s at the 3’ end of DNA
A hydroxyl group
Which part of DNA is hydrophlic?
The sugar-phosphate backbone
Which part of DNA is hydrophobic?
The bases
What is the general method for extracting DNA?
Sample disruption
Release of cell contents
Extraction
Purify and concentrate DNA
What methods of disrupting samples to get DNA are there?
Mechanical
Chemical lysis
Osmotic - put them in pure water and they burst due to osmotic shock
Enzymatic
Lysate
The contents of a lysed cell
Traditional method of extracting DNA
Mix cells with phenol-chloroform solution and centrifuge
Forms solvent and aqueous phase
DNA in aqueous phase
Why does DNA appear in the aqueous phase in phenol-chloroform extraction?
Because DNA is hydrophilic
Ethanol precipitation method of purify and concentrate DNA
DNA is insoluble in ethanol
High speed centrifugation makes a pellet of DNA that can be re-suspended in a small volume of clean H2O
What are the advantages of nucleic acid purification kits?
Allow preparations of high purity
Quick and easy
What are the disadvantages of nucleic acid purification kits?
Expensive
Solutions may be of unknown compisition
What setting on the spectophotometer reads how much DNA is in a sample?
A260
What setting on the spectophotometer reads the purity of DNA in a sample?
A280
What is the optical density to dsDNA (ng/ul) equivalent?
1.0 OD = 50ng/ul
Equation for the concentration of DNA using a spectrophotometer?
DNA conc (ng/ul) = A260 OD x dilution factor x 50
Equation for the purity of DNA using a spectrophotometer?
= A260 / A280
What does a ratio of A260 and A280 at ~1.8 tell us the sample is?
Pure DNA
What does a ratio of A260 and A280 at ~2.0 tell us the sample is?
Pure RNA
What does a ratio of A260 and A280 at <1.6 tell us the sample is?
Other contaminants
Why do A-T base pairs separate at a lower temperature than C-G pairs?
A-T has 1 less H bond
What does the Tm value indicate when heating DNA?
The temperature at which half the base pairs are denatured and half remain intact
If two different samples have different Tm values what does this tell us?
The DNA with the higher Tm value has a greater amount of C-G base pairs
What is reannealing of DNA?
When single strands of DNA find their complimentary strands to reform a double helix
What happens to DNA if you cool it quickly after melting it?
The single strands don’t reanneal
Non-repetitive DNA
Sequences that are unique and occur in only one place in the genome
Repetitive DNA
DNA sequences that are present in more than one copy per genome
Multigene families
Collection of similar genes
Often clustered together
What is a pseugogene?
Genes that have lost protein coding ability
What can cause pseudogenes?
A premature stop codon Insertion Deletion Frameshift mutation Loss of a promotor
Highly repetitive DNA (satellite DNA)
Short sequences repeated many times
Types of middle repettitive DNA
Minisatellites
LINES
SINES
Minisatellites
Variable number of tandem repeats
Sequences about 5-100 bp long
Repeated many times end to end
Varies between individuals
Which type of repetitive DNA is used in DNA fingerprinting?
Minisatellites
LINES
Long interspersed sequences
Up to 6000 bp
SINES
Short interspersed sequences
100-500 bp
Don’t code for anything
Transposons
Jumping genes
Piece of DNA that can move around then genome
What is the cause of repetitive DNA?
Transposons
What enzymes cut DNA?
Restriction enzymes
Why are blunt ends harder to allow annealing than sticky ends?
No H bonds form from overlapping tails of DNA
Makes it harder for DNA ligase to make the phosphodiester bonds
Is DNA positively or negatively charged?
Negatively
How does gel electrophoresis work?
Negative charged DNA moves towards + electrode
Smaller DNA can fit through gaps in gel better than large so travel closer to the + electrode
How does DNA move in gel electrophoresis?
By reptation
What do you use to visualise the DNA in gel elctrophoresis?
Ethidium bromide
What tool is used to compare the movement of the DNA samples in gel electrophoresis?
A DNA ladder
Polyacrylamide gels
Better for smaller fragments of DNA than agarose
Much better resolution - can distinguish difference of 1 bp
How do plasmids behave in agarose?
They can supercoil under the tortional stress
Supercoiled plasmids move move than non-supercoiled as they are more streamlined
Restriction fragment length polymorphisms (RFLP)
Can be used to detect some diseases caused by a single base pair change
How can RFLP be used be used to detect diseases caused by a single base pair change?
Single change causes a loss of a a particular recognition site for an restriction enzyme
When applied the restriction enzyme doesn’t make the cut it usually would in that place
Using gel electrophoresis you can identify the longer than normal length of DNA that wasn’t cut
Name 3 uses of restriction enzymes
Cutting and pasting genes for cloning
Checking whether cloning was successful
Diagnostics
What is the hyperchromic effect?
DNA when heated becomes single stranded
ssDNA is less compact than dsDNA
ssDNA will have a higher A260 reading
Which base pairs separate at a lower temperature?
Why?
A-T
They have 2 H bonds between them while C-G has 3
How is aragose gel made?
Boiled with water then cooled
What are tandem repeats?
Repeated sequences of DNA or RNA that lie next to each other
Satellite DNA
Highly repetitive DNA
Found as clusters of tandem repeats
Permanently coiled tightly into heterchromatin
What does VNTR stand for?
Variable number tandem repeats
Variable number tandem repeats
Short tandem repeats
Fewer copies than satellite DNA
