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MMG 433 EXAM WITH COMPLETE
SOLUTION
Distinguishing benign from pathogenic genetic variation - ANSWER large sample size of sick and healthy patients
Size of human genome - ANSWER 3 billion base pairs
Personalized/precision medicine - ANSWER use genetic info to tailor treatments, anticipate onset of disease, manage health
Hybrid crops - ANSWER superior than either parental lineages; hard to maintain
Challenge with hybrid crops - ANSWER epistatic effects of multiple alleles
Genomics - ANSWER Discipline of mapping, sequencing, analyzing, and comparing genomes; molecular biology, genetics, bioinformatics
Goals of genomics - ANSWER sequence/assemble genomes, identify functional elements, maintenance/evolutionary processes, diversity, expression, phenotypes, altern/design living organisms
Statistical significance - ANSWER calculated as P(mutation | disease)
Confidence that mutation causes disease - ANSWER depends on occurence of that mutation in the general population
Koch's postulates - ANSWER 1. Microbe found in sick, not healthy organsims
- Must be able to isolate from diseased organism
- Microbe causes disease when introduced to healthy organism
- Reisolate from inoculated, diseased nhost
Limitations of Koch's postulates - ANSWER 1. can isolate from healthy & diseased patients
- can't always grow in pure culture
- differeing susceptibilities; can't infect humans
Sequencing by synthesis - ANSWER uses DNA polymerase, sequence template, oligo primer, 4 dNTPs; add one nucleotide type at a time and quantify incorporation into synthesized strand
Sanger-Coulson method - ANSWER 1 or 3 nucleotides added to synthesis reactions to stop synthesis at specific locations; requires 8 reactions and 8 electrophoresis lanes
Minus - ANSWER polymerase stops at missing base
Plus - ANSWER T4 DNA polymerase 3' exonuclease stalled by dNTP
Maxam-Gilbert method - ANSWER ds DNA chemically altered to cleave fragments at specific basis; 4 reactions & 4 electrophoresis lanes required; A+G, G, C, C+T
Problems with Maxam-Gilbert method - ANSWER cumbersome, limited resolution, repeats of same nucleotide difficult to resolve
Sanger Sequencing chain termination - ANSWER dNTPs = DNA elongation, ddNTPs = chain terminators, causes polymerization to stop randomly but at specific base
DNA sequencing by chain-termination - ANSWER family of fragments with different sizes recovered from reaction based on final nucleotide; can view repeats
Polyacrylamide gels - ANSWER can resolve DNA fragment sizes w/ a resolution of a single base; ~200 nucleotides read with high accuracy
Sanger sequencing improvements - ANSWER capillaries allow for faster electrophoresis, smaller footprint, fluorescently labeled ddNTPs allow for laser detection; automate and parallelize for higher throughput; ~1,000 bases
Limitations of Sanger sequencing - ANSWER large amount of template DNA, need flanking DNA of known sequences, expensive ddNTPs, have to sequence 10x to get high confidence
Clone library - ANSWER convenient way to prep template DNA for sequencing
How are clone libraries made? - ANSWER DNA fragments cloned into vectors, transformed into bacterial/yeast hosts to replicate DNA archiving
How are clone libraries used? - ANSWER isolate unique fragments from single colonies and sequence then mapped
EST - ANSWER Expressed Sequence Tag library that contains transcribed genes used to sequence full large genomes
What do mRNAs represent? - ANSWER small fraction of the genome but contain protein coding sequences/introns
3 laws of genetics - ANSWER allele segration, independent assortment, allele dominance
Genetic linkage - ANSWER alleles on the same chromosome are inherited together
Partial linkage - ANSWER some alleles are neither independent nor fully linked;
digestion
FISH (fluorescent in situ hybridization) - ANSWER hybridization of fluorescently labeled DNA probes that can reveal physical locations of specific sequences on chromosome
STS (sequence tagged site mapping) - ANSWER map positions of short DNA sequences unique in the genome using PCR/hybridization
What is needed for STS? - ANSWER collection of overlapping DNA fragments from a single chromosome
How does STS work? - ANSWER if 2 fragments have same STS, then must overlap; if 1 fragment has two different STSs, then those two STSs must be near each other
Shotgun sequencing of small genomes - ANSWER genome assembly from small fragments rely on ability to find overlapping sequences; requiers efficient computational methods
Sequence alignment - ANSWER arranging two or more sequences to identify regions of similarity; identify matches, insertions/deletions, substitutions
Dot plots - ANSWER provide visual overview of possible alignments but do not determine optimal alignment
Needleman-Wunsch algorithm - ANSWER recursive alignment process that aligns each position relative to best alignment; only considers subset of all possible alignments but guarantees to find optimal mathematical solution
Dynamic programming for optimal alignment - ANSWER get alignment with best score; not always biologically meaningful
What determines best scoring alignment? - ANSWER depends on scoring matrix used; calculate statistical significance of alignment score
Sequence homology - ANSWER two similar sequences found in genomes of two different organisms may be related through evolution
Selective pressure - ANSWER random mutations make sequences diverge but selective pressure to preserve function will impose sequence conservation at critical positions
Multiple sequence alignment (MSA) - ANSWER identify conserved sequences across organisms; create evolutionary trees
Problems with MSA - ANSWER computationally expensive to find optimal alignments; have to use heuristic approach that doesn't guarantee optimal alignments
Optimal pairwise alignment - ANSWER to find biologically meaningful alignments, can't use percent identity because not all AA subsitutions are equally likely
P(ab) - ANSWER probability of substitution; determined empirically from trusted set of
aligned sequences
PAM (point accepted mutations) - ANSWER observed mutations in closely related proteins, does not work well for aligning distantly related proteins
BLOSUM (block substitution matrix) - ANSWER observed mutations in distantly related proteins, only use blocks of conserved positions to calculate substitution rates
BLOSUM62 - ANSWER blocks database of trusted alignments that are highly conserved and gap free sequence blocks, keeps alignments with < 62% identity, calculate background frequency of each residue, calculate substitution rate for each residue pair
What does scoring matrix choice depend on? - ANSWER source of sequences to be aligned and evolutionary distance between sequences
What do specialized scoring matrices do? - ANSWER take into account protein structure, develop sequence context to improve alignment accuracy and biological relevance
Common amino acids vs rare amino acids - ANSWER low weights vs high weights
Positive vs negative - ANSWER more likely substitutions vs less likely substitutions
Sequence databases - ANSWER relies on sequence alignments, never perfect because of mutations/sequencing errors, have to return imperfect matches and provide statistical significance
BLAST (Basic Local Alignment Search Tool) - ANSWER compares a sequence of interest with all other DNA or protein sequences deposited in sequence databases; seed and expand alignment approach
Score - ANSWER pairwise alignment score calculated from the scoring matrix (ie BLOSUM62)
Query cover - ANSWER percentage overlap between query sequence and database hit
E-value - ANSWER expected number of hits returned by chance given size of the query seqsuence (false positives)
Max identity - ANSWER number of identical positions between aligned sequences
GenBank - ANSWER NIH database that is an annotated collection of all publicly available DNA sequences
Hamiltonian cycle - ANSWER represent sequencing results as nodes in a graph, connect overlapping sequences with arrow, find the path that travels through every node exactly once and ends at the starting node
Why is the Hamiltonian cycle difficult? - ANSWER requires trillions of pairwise alignments; no efficient algorithm to find it with millions of nodes; feasbile for microbial
What do transcription factors do? - ANSWER control activity of RNA polymerase
Transcription terminator loop - ANSWER formation of hairpin loop made from complementary bases followed by poly-U that destabilizes RNA polymerase; can recognize palindromic sequence
Non-coding tRNA - ANSWER conserved structure of tRNA can be used to identify tRNA genes
Why is it harder to detect genes in eukaryotes? - ANSWER introns interrupt reading frame and can be large, complex promoters, pseudogenes, variable RNA pol/TF binding sies
How can you discover exons and introns? - ANSWER map RNA molecule sequence back to genome sequence; can identify alternative splicing
Problem with RNA seq - ANSWER not all genes transcribed all the time in all tissues
Physical separation of DNA limitation - ANSWER 1 template per reaction, labor intensive to create BAC libraries, fragment can be lost or incompatible with host
Template amplification limitation - ANSWER mistakes in amplification, hard to amplify some regions, need reagents and to know flanking sequences
Sequencing limitations - ANSWER synthesis/sequencing are separate steps, expensive fluorescent terminators, large reaciton volume, limited read length
Template library with water-oil emulsion setup - ANSWER ligate templat to universal primers and attach to beads that are separated using water-oil emulsion, where each droplet contains a bead
Water-oil emulsion sequencing - ANSWER amplify in each droplet, create large library with different fragments in each drop, sequence on glass surface/wells
Benefits of water-oil emulsion - ANSWER no cloning host so don't lose incompatible sequences
Pyrosequencing - ANSWER after a base is added, pyrophosphoric acid is released, used as substrate to generate ATP by sulfurylase, which is used by luciferase to create detectable light
Advantages of pyrosequencing - ANSWER natural nucleotides reduces sequencing errors
Ion torrent - ANSWER release of protons from the addition of a nucleotide can be detected with a voltage meter
Solid phase template amplification - ANSWER attach DNA template to solid surface covered with universal primers; bridge amplification; high density
Cylic reversible termination - ANSWER solid phase chemiluminescent signal not strong enough to detect, determine sequence using reversible blocking group that allows elongation after signal detection
3' blocked reversible terminators - ANSWER unincorporated nucleotides washed away, 4 color imaging acquired, cleavage step removes terminating group and fluorescent to restore 3'-OH
How are solid phase sequences read? - ANSWER scan glass slide after each cycle of fluorescent terminator addition which creates light dot; short reads because of errors
Single molecule real time long read sequencing - ANSWER RNA pol anchored to well, sequences phospholinked hexaphosphate nucleotides, read fluorescent intensity
What is machine learning used for? - ANSWER annotate functional elements, generate new testable hypotheses, combine to build predicrive models
Supervised learning - ANSWER requires a set of known examples (training set) to build a predictive model, which is used to make predictions on a new data set (testing set); can continue cycle to improve
Example of supervised learning - ANSWER created a position frequency matrix from an alignment of promoter sequences to determine probability that a sequence is a promoter
Unsupervised learning - ANSWER discover structures unlikely to appear by chance; may reveal undiscovered biological functions
Iterative improvement of models - ANSWER train model/discover unlikely signal, predict or generate new hypotheses, validate predictions and use them to continue to train model
Generative model - ANSWER build from all features characterizing a class (Hidden Markov, Position Frequency Matrix), interpretable description of class and quantifiable variablity; generates example; requires more training
Discriminiative models - ANSWER focuses on distinctive features, more efficient but not uesful when context changes
Precision - ANSWER true positive / (true positive + false positive); how many selected items are actually the item of interest
Sensitivity (recall) - ANSWER true positive / (true positive + false negative); how many of the relevant items are selected
Specificity - ANSWER true negative / (true negative + false positive); how many of non-relevant items are actually selected
Accuracy - ANSWER true positive + true negative / everything; how often is the model actually correct
Precision recall curve - ANSWER tradeoff between specificity and sensitivity; high specificity means few false positives, high sensitivity means few false negatives
Genome replication in bacteria - ANSWER signle origin of replication, proceeds in two forks in opposite direction, termination happens when ter elements trap replication forks
DNA pol III - ANSWER main polymerase for DNA synthesis
DNA pol I and II - ANSWER mainly used for DNA repair
Origin of replication - ANSWER cluster of DnaA binding sites
DnaA - ANSWER protein that binds and loops genomic DNA to promote unwinding at AT rich region
DnaB - ANSWER helicase binds melted region with the help of DnaC to initate genome replication
RNA primase - ANSWER make RNA primer for DNA to extend
DNA polymerase I - ANSWER excise RNA primer, fills gaps to synthesize remaining DNA
Tus protein - ANSWER binds ter sequences, inhibits helicase activity when polymerase hits site facing the opposite direction and stops replication
leads to aging/cell death
Telomerase enzyme - ANSWER contains catalytic built in RNA template, attaches to 3' end of chromosome and elongates
Why are telomeres repeated sequences? - ANSWER easy to create repeated short sequence with telomerase
G1 stage - ANSWER initiate replication at multiple locations
S phase - ANSWER replication forks move outward and replicate genome
G2 phase - ANSWER fully replicate genome, prep for mitosis
How where yeast cells synchronized? - ANSWER checkpoint deficient mutants, limited DNA replication to regions close to origin of replication
Marker frequency analysis - ANSWER quantify number of copies in a cell; localize origin of replications and calculate speed of replication forks along the chromosome
How to determine copy number of each genomic region - ANSWER count the number of times fragments were sequenced with shotgun sequencing and mapping approach; should see regions close to origin replicated 2x as often
Why normalize marker frequencies against non-replicating cells? - ANSWER raw sequence efficiency not equal throughout genome; some sequences are more difficult to read
What does in peak mean in relative copy number profile - ANSWER corresponds to number of times the fragment was seen; means replication started there
How can dynamics of replication be characterized? - ANSWER time course experiment on synchronized cells, look at relative copy number to determine time it takes to replicate
Asynchronous experiment - ANSWER genomic regions close to the origin of replications have copy number higher than regions further away
Where do more errors occur? - ANSWER lagging DNA strand; more complex
DNA polymerase error rate - ANSWER 1 error in 10^7 nucleotdies; overall rate reduced to 1 in 10^10 because of repair mechanisms (1 in every 2000 genome copies)
Transition - ANSWER purine to purine (A to G) or pyrimidine to pyrimidine (C to T)
Transversion - ANSWER Purine to pyrimidine (A/G to C/T)
When is a mutation propagated? - ANSWER when the error is replicated
What happens when bases are mismatched? - ANSWER polymerase is slowed, exonuclease is faster (normally it is the opposite)
Proof reading - ANSWER competition beteween polymerase and exonuclease; when mismatch occurs, polymerase activity reduced, mismatched nucleotide excised
Alternative nucleotide tautomers - ANSWER normally biased towards correct structural isoform, but occasional shift in replication causes incorporation of wrong nucleotide
Base analogs - ANSWER incorporated into DNA and cause mutations; 5-bromouracil
Enol form of thymine - ANSWER base pairs G not A
detected by protein complex; recongition of stalled RNA pol at damaged nucleotides
How does excision machinery work? - ANSWER entire segment of a dozen nucleotides containing damaged nucleotides excised and resynthesized using DNA pol and ligase
Most common mechanism to repair damaged DNA - ANSWER excision repair
DNA adenine methylase (Dam) - ANSWER adds methyl group to adenine bases at GATC site
Strand specific mismatch repair - ANSWER repair machinery uses methylation patterns to distinguish newly synthesized strand (won't have methyl group) from old template, mismatches repaired on new strand
Double strand breaks - ANSWER most catastrophic, difficult to repair
Repairing double strand breaks - ANSWER can rejoin directly, but leads to major chromosomal rearrangements if there are multiple breaks
Homologous recombination - ANSWER if multiple copies of the genome are available, repair breaks with this method, single strand of non-damaged DNA invades double strand break and allows replication to occur
What does homologous recombination often lead to? - ANSWER resolution of branch migration can lead to a crossover
Denococcus radiodurans - ANSWER one of the most radiation resistant organisms; efficient DNA repair; each cell has 4 copies of genome in stationary phase; 8-10 during replication
How does D. radiodurans reconnect chromosome fragments? - ANSWER single strand
annealing; homologous recombination to reconstrcut full genome from multiple pieces
What is needed to map double strand breaks into genome? - ANSWER proximal (linked to biotin) and distal DNA linkers w/ hairpin structures, barcodes, deep sequencing
Aphicolidin - ANSWER DNA polymerase inhibitor that stalls replication fork, increases DSBs
Neocarzinostatin - ANSWER induces DSBs directly, more randomly
What did mapping of DSBs show? - ANSWER distribution not uniform in genome
Where are DSBs enriched? - ANSWER satellite repeats that form hairpins, pericentromeric and centromeric regions; transcribed regions with high density protein coding
Autopolyploidy - ANSWER more than two sets of chromosomes; common in plants
What does autopolyploidy allow? - ANSWER potential for gene expansion/diversification because extra genes can get mtuations without strong selective pressure
Allopolyploidy - ANSWER interbreeding between two different species with viable hybrid
What can homologous DNA recombination cause? - ANSWER generate large genome rearrangements, fragment losses, sequence duplications
Segmental duplication - ANSWER homologous/non-homologous recombination underlie large chromosomal rearragnements that cause gene duplication/deletion/fusion
Unequal crossing over - ANSWER repeat sequences in homologous chromosome pairs
Gene transfer agents - ANSWER prophages under control of host, transfer random pieces of genome
Intracellular transfer - ANSWER endosymbiont can exchange genetic material with eukaryotic hosts; mitochondria and plastids, gene transfer through phagotrophy
Web of Life - ANSWER ubiquity of HGT in microbes makes it difficult to determine lineages
How to detect HGT - ANSWER disagreements between tree structures
Reference tree construction - ANSWER use genes unlikely to be transferred (ribosomal proteins, core processes), multi-locus sequence analysis
Pathogenicity islands - ANSWER large genomic regions that contain genes involved in the synthesis of virulence factors; probably acquired by HGT; found in pathogenic strains of species
Sequence characteristic evidence for HGT - ANSWER GC content, codon use, regions that are different content probably came from somewhere else
HGT frequency - ANSWER occur more frequently between closely related organisms; sequence similarity promotes homologous recombination
Genome reduction in symbiotic bacteria - ANSWER live in stable/predictable conditions, need fewer functions, without selective pressure, lose genes through large chromosomal rearrangements
Core genome - ANSWER gene families present in all genomes of a group
Pan genome - ANSWER collection of all genes present in members of a group; shows
wide range of biological function; represents set of genes potentially available via HGT
Accessory genome - ANSWER genes present in only one or a few members
Original phylogeny basis - ANSWER look at physical features, limited to what you can see, difficult/impossible for microbes
Molecular phylogeny - ANSWER look at rRNA, use sequence information, each position in the gene is a trait
Phylogenetic tree inference - ANSWER comes from available data and assumptions about the mechanisms of evolution; many possible different trees
Maximum parsimony - ANSWER minimizes number of changes to explain evolution, no evolutionary basis
Drawbacks to maximum parsimony - ANSWER does not correct for multiple substitutions at the same site, sequence evolution not always parsimonious
Distance based phylogeny - ANSWER calculate distances between sequences, create matrix, construct tree based on pairwise distances, dependent on data scoring
Neighbor-joining method - ANSWER find nodes close to each other, far from everyone else, join together and recalculate distance to remaining nodes
Agglomerative clustering method - ANSWER look at matrix, find smallest distance between neighbors and link
Benefits of neighbor-joining method - ANSWER computationally efficient, useful for large datasets
