module 3 Flashcards
what are nucleic acids?
chains of nucleotides, self assembly with weak forces, base pairing into helical structure
oligonucleotides: 50 or fewer
polynucleotides: more than 50
what does a nucleotide consist of?
heterocyclic base, five-carbon sugar (pentose), and a phosphate group
what is chargaff’s rule?
the number of A residues equals the number of T residues
and
the number of G residues equals the number of C residues
important for information storage and replication
what is hydrogen bonding in dna?
base paring between nitrogenous bases, CG has 3 bonds and AT has 2 bonds
what is x ray diffraction?
analyze dna fibres to produce diffraction pattern of spaced atoms of a crystal, determines arrangment of atoms
what did watson and crick discover?
helical periodicities of 3.4Å and 34Å, 10.5 base pairs in each complete turn of the double helix
why is hydrophobic stacking important?
energetically favourable, stabilizes helix, minimizes contact of hydrophobic bases with water
what are the three functions of dna?
1.Long-term storage of genetic information
- Acting as a template for dna replication
- Coding for proteins
what are the four internal forces of dna stability?
- hydrophobic interactions
- van der waals interactions
- hydrogen bonds
- ionic interactions (backbone)
what are the four external forces of dna stability?
- temperature
- salt
- proteins
- organic solvents
what is coding rna?
mrna carries genetic information from nucleus to ribosomes where protein is produced
what is noncoding rna?
trna, rrna, lncrna, snrna, mirna, sirna, snorna, and catalytic rna
what is noncoding rna?
trna, rrna, lncrna, snrna, mirna, sirna, snorna, and catalytic rna
what are three examples of secondary structures?
- helical structures
- internal loops
- hairpin loops
what is quantification?
determines the concentration of dna and rna in a sample
what is uv absorption?
amount of light absorbed is proportional to the amount of protein and/or nucleic acid present
what is beer’s law?
absorbance of light at a certain wavelength is directly proportional to the concentration of the solution (darker=more absorbing)
what is optical density?
amount of UV light able to pass through a solution
what is the different between hypo- and hyper- chromicity?
hypo: large decrease in light absorption at 260 nm as dna forms
hyper: large increase in light absorption at 260 nm as dna unwinds
what is denaturation?
when exposed to extreme ph or temperature, viscosity decreases and dna strands break apart
what is stingency of dna hybridization? high vs low?
hybridization can occur between two non-complementary strands of nucleic acids
high: two strands are compatible
low: base mismatches
what increases dna hybridization?
formation is not favoured with increase: high temp, low salt, and presence of organic solvents
what are the four steps of PCR?
- denaturation (94-95)
- annealing (50-56)
- elongation (72)
- amplification
- repeated 25 to 30 times over a few hours
why are pcr primers used? (applications)
dna samples from human remains can trace evolution of pathogenic viruses to detect infections
what is the design of a pcr primer?
complementary to seqeunce, 18-25 nucleotides and 40-60% GC content
what is gel electrophoresis?
separates mixtures of large charged molecules, based on size
1. gel matrix is composed of agarose, does not disrupt base pairs
2. voltage is applied, dna/rna migrate towards positive end of the gel
3. larger molecules move more slowly, retain closer to top
what is the use of ethidium bromide?
inserts between nucleic acid stands and fluoresces when exposed to UV light and detects nucleic acids in a gel
function of reverse transcriptase?
amplifies and sequences gene segments without introns
what is SYBR green?
fluorescent dye added to reaction mix and is brighter when bound to dsdna
what are the four parts of qPCR?
- exponential phase
- plateau phase
- cycle threshold
- ct value
what is sanger sequencing? (purpose)
dideoxy chain-termination method, enyzmatic synthesis of dna strand, catalyzed by polymerase
what are the 6 steps of sanger sequencing?
- dna denaturation: heat denatures dsdna to ssdna forming template and complementary strands
- primer: anneals to template, adds nucleotides
- free nucleotides: dATP, dCTP, dGTP, dTTP added
- modified nucleotides: dideoxy nucleotide
- chain termination: lack 3’-OH of phosphodiester bond causing cease of extension
- gel electrophoresis: sample is collected and mixture is added to be detected by autoradiography
what is dye-terminator sanger sequencing?
ddNTPs of colours are added to same reaction mixture, products are excited by laser detected one nucleotide at a time
what is in vitro pcr amplification?
synthetic adapters are ligated at ends to serve primer binding regions, enables PCR amplification
what is in vivo dna replication?
incorporates segment into vector and replicates as bacteria, enables replication amplification
what are the five steps of molecular cloning?
- endonuclease cleaves dna into smaller fragments
- cloning vectors carrier dna, self-replication (plasmid vectors)
- joining two dna fragments covalently with ligase (vector to fragment)
- move recombinant dna from tube to host (for enzymatic machinery)
- cloning vector allows cell to survive
what is ori?
origin of replication initiated by cellular enzymes
what are restriction sequences?
provide sites where the plasmid can cut into and insert foreign dna
what does antibiotic resistance do?
allows selection of cells that contain intact plasmid or recombinant version using antibiotics (tetracycline and ampicillin)
three limitations of sanger sequencing?
- slow and expensive
- read lengths are 1000-1500 bases
- large segments must be broken down and analyzed one at a time
what is the purpose of next generation sequencing?
allows rapid sequencing of large dna segments into smaller fragments
what is reversible terminator sequencing?
nucleotides are labeled with fluorescent tags bound to modified nucleotides using a cleavable linker region
what are the three parts to adaptor ligation?
- terminal sequences: essential for cluster generation
- index sequences: unique barcode for dna fragments
- allows pairing from 3’ and 5’ ends
what are the four steps of cluster generation?
- library is added to flow cell, terminal sequences hybridize with oligonucleotides
- og template is washed away, dna covalently bound to flow cell
- adaptor sequence 3’ hybridizes with oligonucleotide, two strands are denatured
- repeated, reverse strands are hydrolyzed and washed away, leaves cluster of unidirectional clonal strands
three characteristics of the human genome?
- 3 billion nucleotide base pairs
-23 pairs of chromosomes
-20,000-25,000 genes
what is single nucleotide polymorphisms?
represents base pair change to distinguish one species from another
what is large genomic rearrangments?
inversions as a result of segment duplication leading to mutation/fushion of dna to form a hybrid gene
what are homologs?
two genes with sequence similarity implying evolutionary relationship
what are orthologs?
sequence and functional relationship (mice and humans have leptin gene with the same function)
what are paralogs?
sequence and functional relationship, arose from gene duplication in a single species (ins1/ins2 in mice)
what are three purposes for chromosome packaging?
-highly organized
-allow access to factors that regulate dna replication
-allow access to factors that regulate transcription
what are the levels of organization? (disordered to ordered)
nucleotides
dna double helix
histones
nucleosomes
chromatin
chromosome
what are histones?
large protein of chromotin, positively charged, assembly of octamers, dna wraps around with negatively charged backbone, forms nucleosome, highly conserved
what is the purpose of H1?
stabilize nucleosomes, promotes higher order chromosme structure, enhancse repression of transcription
what are chromatin remodeling complexes?
repositions nucleosome to different location, ejects nucleosome from dna, replaces with one histone variant (requires atp, atpase domain)
what are the two histone modifying enzymes?
covalently modify n-terminal tails of histone proteins
cis: modify histone for open/close of chromatin or tightening/loosening of nucleosomes
trans: attract proteins producing chromatin change
what are the three H2A variants?
H2AX: dna repair (attracts proteins) and genetic recombination
H2AZ: stabilize open state of chromatin
macroH2A: X-chromosome inactivation
what are the two H3 variants?
H3.3: stabilizes open state of chromatin
CENPA: contains extension to connect kinetochore
what are HDACs?
deacetylases that remove acetyl groups from histones
what are HATs?
acetyltransferases that add acetyl groups to histones
what are HMTs?
methyltransferases that add methyl groups to histones
what is the jumonji family (KDMs)?
demthylases that remove methyl groups from histones
what are the four steps to chromatin immunoprecipitation (ChIP)?
- cells treated with formaldehyde covalently crosslink nucleosome to dna
- antibody to specifc histon is used to immunoprecipitate the complex, dna not bound is washed away
- crosslinks are reversed by heating, regions are amplified by pcr/qpcr
- released dna is labeled and probes a microarray of sequences associated with nucleosomes (ChIP experiment)
what are bromodomains?
proteins containing bromodomain recognize acetylated nucleosomes, stabilizing the open/closed chromatin state
what are chromodomains?
proteins containing chromodomains recognize methylated nucleosomes, stabilizing closed/inactive chromatin state
