MMG 433 Final Exam Study Guide With
Correct Solution
Flux balance analysis (FBA) - ANSWER a mathematical approach for analyzing the flow
of metabolites through a metabolic network, thereby making it possible to predict the
growth rate of an organism or the rate of production of a biotechnologically important
metabolite. These network reconstructions contain all the known metabolic reactions in
an organism and the genes that encode each enzyme.
Genome-scale metabolic network - ANSWER Discrepancies between predictions and
experiments can help generate testable hypotheses about missing pathways and
improve gene functional annotations
FBA can be used to predict the effects of gene deletions. Can measure input and output
to determine what the cell is doing.
FBA relies on knowledge of - ANSWER the full metabolic network
Predicting metabolic fluxes - aerobic glucose respiration - ANSWER uses oxidative
phosphorylation pathways - no output from fermentation output
Predicting metabolic fluxes - anaerobic glucose fermentation - ANSWER uses
fermentation pathway - no output from oxidative phosphorylation pathway product
Multiplex automated genome engineering (MAGE) - ANSWER MAGE allows the
production of multiple targeted mutations through iterative oligonucleotide mediated
allelic replacement
What happens during MAGE? - ANSWER ssDNA anneals to the lagging strand of the
replication fork between Okazaki fragments.
After 2 rounds of genome replication, a fully mutated genome is created.
Different types of small mutations can be introduced anywhere in the genome.
MAGE works well for - ANSWER small targeted mutations - not good for full gene
mutations
The MAGE workflow - ANSWER 1 - Goal
2 - ID target loci
3 - Design oligos
4 - Predict cycle time
,5 - MAGE cycling
6 - Screen/selection - screen for colonies that have mutations of interest
Expanding the genetic code in E. coli - ANSWER MAGE was used to remove all 321 TAG
trinucleotides (which encode the amber stop codon) from E. coli DNA (by converting
them to TAA, which encodes the ochre stop codon) as well as the release factor that
recognizes them.
This procedure yielded a blank codon, which was then reassigned to encode a
non-standard amino acid.
CRISPR - ANSWER uses Cas9 for editing genomes
Cas9 - ANSWER An endonuclease that cuts 2 strands
Can be expressed in all kinds of cells - can edit any genome we want
Can target any sequence in the genome
Alternative uses of Cas9 - ANSWER Broken scissors - Cas9 enzyme can be broken so
that it no longer cuts DNA. But with the right guide RNA, it can still attach to specific
parts of the genome. Becomes DNA-binding protein and can become a repressor.
CRISPR epigenetics - add domains that will affect DNA/ change activity of gene
Designing genomes - ANSWER Make genome from scratch
natural cell → genomic DNA → Sequence → DNA sequence database → Genome design
→ DNA synthesis → Genome assembly in yeast → Synthetic genome → Transplant to
recipient cell → Synthetic cell
Gibson assembly and yeast assembly - ANSWER how to stitch fragments together
One step isothermal in vitro reaction to assemble up to 5 overlapping DNA fragments
using 3 enzymes:
1. 5'-exonuclease
2. DNA polymerase
3. DNA ligase
Yeast have an efficient homologous recombination system - transform 25 overlapping
DNA fragments into yeast, yeast will assemble into a circular genome.
Synthetic Yeast III chromosome - ANSWER Step 1: Synthesize building blocks (BBs)
from oligonucleotides
Step 2: Assemble 2-4 kb minichunks
,Step 3: Replace native III with minichunks
What percent of the Human genome is unclassified? - ANSWER 23.6%
How many protein encoding genes are in the human genome? - ANSWER ~21,000
Types of Genome Maps - ANSWER Genetic, Cytological, Physical, Other
Genetic Genome Maps - ANSWER Measures distances between genes by the
recombination frequency between mendelian gene markers
Cytological Genome Maps - ANSWER Measures distances between visible bands or
strained regions found in chromosome karotypes
Physical Genome Maps - ANSWER Measures distances between genetic elements in
terms of length of DNA between them → mainly by sequencing
Ideograms - ANSWER Cartoon images of banded chromosomes
How to do cytological maps - ANSWER 1. Chromosome spread - capture cell during
mitosis when condensed, use saline to inflate chromosome.
2. Sort chromosomes by size to get karotype
What are banding patterns caused by? - ANSWER Non-uniform distribution of GC and
AT content
The most commonly used stain to view banding patterns of stained metaphase
eukaryotic chromosomes is? - ANSWER Glemsa dark, well-staining regions are AT-rich,
light regions are GC rich
What are banding patterns useful for? - ANSWER Distinguishing chromosomes of similar
size, and for identifying cytogenetic abnormalities such as translocations
Short arm of chromosome is called - ANSWER p telomere
Long arm of chromosome is called - ANSWER q telomere
Fluorescent In Situ Hybridization (FISH) - ANSWER -FISH can be used to locate large
pieces of DNA to withing 5-10 Mb on a chromosome
-A probe can be labeled with a fluorescent dye
-The probe can be hybridized to a prepared metaphase chromosome spread
-Multiple probes with different colored dyes can be used to map multiple clones at the
same time
How Chromosome Paints are Produced - ANSWER "Paints" for individual chromosomes
are produced by "fluorescent activated cell sorting (FACs)". The size of the
chromosome determines the amount of EtBr bound. The computer uses a charged plate
, to deflect the charged droplet into an individual tube. After the EtBr is removed, each
pool of sorted chromosomes is then labeled with a different color fluorescent dye
Chromosome painting involves - ANSWER the generation of purified DNA probe
collections for individual chromosomes. Each chromosome probe sample is
differentially labeled with mixed fluorescent labels whose hybridization can be sorted
out by computer and given a pseudo-color image.
array comparative genome hybridization (aCGH) - ANSWER put test DNA and reference
DNA, green wells shows that there is a gain in test DNA, red shows that there is a loss in
test DNA. there is then a high-density tiling array.
ROMA - ANSWER representational oligonucleotide microarray analysis - covers whole
genome but at a lower density of data points -less expensive
part of aCGH
Three most commonly used genome browsers - ANSWER NCBI
Ensembl
USCS →90% of scientists use
Differences in genome size between prokaryotes and eukaryotes - ANSWER ~90% of
eubacteria and archaea genomes code for proteins; only 1.5% (human) to 10% (yeast) of
eukaryotic genomes code for proteins
Noncoding eukaryotic DNA is primarily
-intergenic DNA
intronic DNA
-a minor proportion of all genes encode RNA species as the final product
Another small portion of the non-coding DNA is regulatory in nature. The ration of
regulatory to coding DNa is thought to be much greater in higher eukaryotes compared
to eubacteria or archaea because of the greater cellular complexity
Eukaryotic Gene Nomenclature - ANSWER -Human gene designations are given in all
capital letters and arabic numbers
-Official symbols are cleared by the HUGO Nomenclature committee
-Rodents have gene symbols that start with a capital letter, but with the remaining
letters being lower case
-Researchers of most non-rodent vertebrate genomes follow human nomenclature as
closely as possible
-roughly 95% of genes in one vertebrate have orthologs in other vertebrates
Correct Solution
Flux balance analysis (FBA) - ANSWER a mathematical approach for analyzing the flow
of metabolites through a metabolic network, thereby making it possible to predict the
growth rate of an organism or the rate of production of a biotechnologically important
metabolite. These network reconstructions contain all the known metabolic reactions in
an organism and the genes that encode each enzyme.
Genome-scale metabolic network - ANSWER Discrepancies between predictions and
experiments can help generate testable hypotheses about missing pathways and
improve gene functional annotations
FBA can be used to predict the effects of gene deletions. Can measure input and output
to determine what the cell is doing.
FBA relies on knowledge of - ANSWER the full metabolic network
Predicting metabolic fluxes - aerobic glucose respiration - ANSWER uses oxidative
phosphorylation pathways - no output from fermentation output
Predicting metabolic fluxes - anaerobic glucose fermentation - ANSWER uses
fermentation pathway - no output from oxidative phosphorylation pathway product
Multiplex automated genome engineering (MAGE) - ANSWER MAGE allows the
production of multiple targeted mutations through iterative oligonucleotide mediated
allelic replacement
What happens during MAGE? - ANSWER ssDNA anneals to the lagging strand of the
replication fork between Okazaki fragments.
After 2 rounds of genome replication, a fully mutated genome is created.
Different types of small mutations can be introduced anywhere in the genome.
MAGE works well for - ANSWER small targeted mutations - not good for full gene
mutations
The MAGE workflow - ANSWER 1 - Goal
2 - ID target loci
3 - Design oligos
4 - Predict cycle time
,5 - MAGE cycling
6 - Screen/selection - screen for colonies that have mutations of interest
Expanding the genetic code in E. coli - ANSWER MAGE was used to remove all 321 TAG
trinucleotides (which encode the amber stop codon) from E. coli DNA (by converting
them to TAA, which encodes the ochre stop codon) as well as the release factor that
recognizes them.
This procedure yielded a blank codon, which was then reassigned to encode a
non-standard amino acid.
CRISPR - ANSWER uses Cas9 for editing genomes
Cas9 - ANSWER An endonuclease that cuts 2 strands
Can be expressed in all kinds of cells - can edit any genome we want
Can target any sequence in the genome
Alternative uses of Cas9 - ANSWER Broken scissors - Cas9 enzyme can be broken so
that it no longer cuts DNA. But with the right guide RNA, it can still attach to specific
parts of the genome. Becomes DNA-binding protein and can become a repressor.
CRISPR epigenetics - add domains that will affect DNA/ change activity of gene
Designing genomes - ANSWER Make genome from scratch
natural cell → genomic DNA → Sequence → DNA sequence database → Genome design
→ DNA synthesis → Genome assembly in yeast → Synthetic genome → Transplant to
recipient cell → Synthetic cell
Gibson assembly and yeast assembly - ANSWER how to stitch fragments together
One step isothermal in vitro reaction to assemble up to 5 overlapping DNA fragments
using 3 enzymes:
1. 5'-exonuclease
2. DNA polymerase
3. DNA ligase
Yeast have an efficient homologous recombination system - transform 25 overlapping
DNA fragments into yeast, yeast will assemble into a circular genome.
Synthetic Yeast III chromosome - ANSWER Step 1: Synthesize building blocks (BBs)
from oligonucleotides
Step 2: Assemble 2-4 kb minichunks
,Step 3: Replace native III with minichunks
What percent of the Human genome is unclassified? - ANSWER 23.6%
How many protein encoding genes are in the human genome? - ANSWER ~21,000
Types of Genome Maps - ANSWER Genetic, Cytological, Physical, Other
Genetic Genome Maps - ANSWER Measures distances between genes by the
recombination frequency between mendelian gene markers
Cytological Genome Maps - ANSWER Measures distances between visible bands or
strained regions found in chromosome karotypes
Physical Genome Maps - ANSWER Measures distances between genetic elements in
terms of length of DNA between them → mainly by sequencing
Ideograms - ANSWER Cartoon images of banded chromosomes
How to do cytological maps - ANSWER 1. Chromosome spread - capture cell during
mitosis when condensed, use saline to inflate chromosome.
2. Sort chromosomes by size to get karotype
What are banding patterns caused by? - ANSWER Non-uniform distribution of GC and
AT content
The most commonly used stain to view banding patterns of stained metaphase
eukaryotic chromosomes is? - ANSWER Glemsa dark, well-staining regions are AT-rich,
light regions are GC rich
What are banding patterns useful for? - ANSWER Distinguishing chromosomes of similar
size, and for identifying cytogenetic abnormalities such as translocations
Short arm of chromosome is called - ANSWER p telomere
Long arm of chromosome is called - ANSWER q telomere
Fluorescent In Situ Hybridization (FISH) - ANSWER -FISH can be used to locate large
pieces of DNA to withing 5-10 Mb on a chromosome
-A probe can be labeled with a fluorescent dye
-The probe can be hybridized to a prepared metaphase chromosome spread
-Multiple probes with different colored dyes can be used to map multiple clones at the
same time
How Chromosome Paints are Produced - ANSWER "Paints" for individual chromosomes
are produced by "fluorescent activated cell sorting (FACs)". The size of the
chromosome determines the amount of EtBr bound. The computer uses a charged plate
, to deflect the charged droplet into an individual tube. After the EtBr is removed, each
pool of sorted chromosomes is then labeled with a different color fluorescent dye
Chromosome painting involves - ANSWER the generation of purified DNA probe
collections for individual chromosomes. Each chromosome probe sample is
differentially labeled with mixed fluorescent labels whose hybridization can be sorted
out by computer and given a pseudo-color image.
array comparative genome hybridization (aCGH) - ANSWER put test DNA and reference
DNA, green wells shows that there is a gain in test DNA, red shows that there is a loss in
test DNA. there is then a high-density tiling array.
ROMA - ANSWER representational oligonucleotide microarray analysis - covers whole
genome but at a lower density of data points -less expensive
part of aCGH
Three most commonly used genome browsers - ANSWER NCBI
Ensembl
USCS →90% of scientists use
Differences in genome size between prokaryotes and eukaryotes - ANSWER ~90% of
eubacteria and archaea genomes code for proteins; only 1.5% (human) to 10% (yeast) of
eukaryotic genomes code for proteins
Noncoding eukaryotic DNA is primarily
-intergenic DNA
intronic DNA
-a minor proportion of all genes encode RNA species as the final product
Another small portion of the non-coding DNA is regulatory in nature. The ration of
regulatory to coding DNa is thought to be much greater in higher eukaryotes compared
to eubacteria or archaea because of the greater cellular complexity
Eukaryotic Gene Nomenclature - ANSWER -Human gene designations are given in all
capital letters and arabic numbers
-Official symbols are cleared by the HUGO Nomenclature committee
-Rodents have gene symbols that start with a capital letter, but with the remaining
letters being lower case
-Researchers of most non-rodent vertebrate genomes follow human nomenclature as
closely as possible
-roughly 95% of genes in one vertebrate have orthologs in other vertebrates
