Chromosome structure Flashcards
Function of genetic material
must replicate, control growth & development of the organism, and allow the organism to adapt to changes in the environment
Griffiths experiment
virulent bacteria kill mice, non virulent bacteria do not. Heat killed virulent bacteria do not kill mice but mixture of heat killed virulent and living non virulent kill the mouse
Transformation occurred - live, non-virulent strain picked up free DNA from dead virulent strain
Avery MacLeod McCarty experiment
using same heat killed virulent and live non-virulent strains, they treated mixtures of both with an RNAse, a protease and a DNAse (respectively)
RNAse and protease yielded virulent bacteria where the DNAse did not because it broke down the free DNA in the solution so the non-virulent bacteria could not take it up
In some viruses the genetic info is in the RNA. True or false
TRUE
3 subunits of DNA
the sugar (pentose), phosphate group and nitrogenous base
4 nitrogenous bases
2 purines (adenine and guanine) and 2 pyrimidines (cytosine & thymine)
How can DNA and RNA be differentiated
Ribose has an OH at the 2 carbon where deoxyribose only has a H
Nucleotides are linked by a phosphodiester bond, which is?
Links 3’ C of one sugar to 5’ C of another
Chargaffs rule
amount of purines = amount of pyrimidines (%A = %T, %G = %C) in organisms
Adenine and thymine are complementary, guanine and cytosine are complementary
A and T form 2 H bonds and G and C form 3 H bonds
William Atsbury found that DNA was a polymer of stacked bases. True or false?
TRUe
Rosalind Franklin took an X-ray crystallography picture of DNA, showing that it is a double helix, what did this show?
Two strands have opposite chemical polarity (3 to 5)
Molecular hybridization
a probe binds to genetic material and acts as a beacon to indicate where a chromosome, gene, or RNA molecule is in a cell
Gel Electrophoresis
DNA loaded into gel made of polyacrylamide or agar and migrate across agar based on charge (towards positive pole), larger samples stop sooner, smaller samples go far
Restriction Enzymes (RE)
group of endonucleases produced in bacteria as a means of destroying foreign DNA, cleave DNA at restriction sites
Cloning plasmids have 3 identifiable markers, what are they
MCS, ORI (origin of replication which enables plasmid replication in host cells), and a selectable marker (e.g. antibiotic resistance)
Multiple Cloning Site (MCS)
region with many restriction sites into which exogenous DNA is inserted (in a bacterial plasmid usually)
Digestion, ligation, transformation, selection
plasmid & foreign DNA are cleaved with RE forming sticky ends
DNA fragments & plasmid hybridize at sticky ends. DNA ligase forms phosphodiester bonds to seal nicks in each strands
ligated plasmid is mixed with bacterial cells under conditions to optimize transformation. Plasmid replicates within host cells as cellular machinery recognizes the ORI within the plasmid.
only cells containing the plasmid will grow on antibiotic-containing plates (in this case, ampicillin), forming colonies which all have the plasmid.
Polymerase Chain Reaction (PCR)
Denaturation -DNA double strand is separated by 95ºC temperature
Primer annealing- specific primers engineered that are complementary to the 2 strands
DNA replicates 5’ to 3’ so primers need to be specific in direction & order
Forward primer - anneals to bottom strand and has same sequence as top strand
Backward primer - reverse of above
Elongation - DNA polymerase duplicated template DNA at 72ºC
72ºC is still quite high, that’s why Taq DNA polymerase (enzyme from thermophilic bacteria) is used for this
At every round, amount of DNA doubles - 2, 4, 8, 16, 32, etc
Next Generation Sequencing
genomic DNA to be sequenced is first fragmented, then adaptor sequences are added to each fragment, then fixed to solid surface (microchip or silicone bead)
PCR is then used to amplify each fragment so that each bead contains thousands of copies of the same fragment
Super-coiled (chromosomes)
multiple rotations of circular DNA (think of twisting an elastic) which allows it to become very compacted. (with the help of RNA and proteins)
Haploid DNA of humans is 3.3 x 10^9 nucleotides (1000x larger than bacteria)
true or false
TRUEE
A chromosome is one long linear molecule of DNA and it contains 2 types of proteins, which are?
Histones and non-histone proteins, since histones are positively charge they attract negative DNA
Chromatin
DNA + histones + protein
How many levels of organization does chromatin have
3
Nucleosomes (level of organization)
core is octamer of histones, 146 bp of DNA wrapped, 1.75 turns/nucleosome
Linker DNA (between nucleosomes) varies in length from 8-114 bp
Nucleosomes are 11 nm wide - must take up more space but more material is fit in
Essentially a greater efficiency of space than small but spread out uncoiled DNA
30 nm chromatin fibre (organization)
less elegant compaction, just scrunching of nucleosomes
Interphase chromosomes (organization)
DNA is tethered to non-histone protein scaffold
Structure is 300 nm overall until chromosome goes into metaphase chromosome
