Chapter 9: DNA-Based Information Technologies Flashcards
genome
the complete haploid genetic complement of an organism
genomics
the study of DNA on a cellular scale (contributes to systems biology)
clone
an identical copy
DNA cloning
selective amplification of a particular gene or DNA segment so that its genetic information may be studied and utilized
recombinant DNA technology or genetic engineering
the methods used to accomplish DNA cloning and related tasks
the process of DNA cloning involves…
generating a recombinant vector
cloning vectors
small DNAs capable of autonomous replication
based off of plasmids- able to replicate themselves and propagate
example: bacteria, covid
recombinant DNAs
composite DNA molecules comprised of covalently linked segments form 2+ sources
advantages of cloning in E.coli
-DNA metabolism is well understood
-many naturally occurring cloning vectors (plasmids and bacteriophages)
-techniques for moving DNA from one bacterial cell to another
What enzymes are used to yield recombinant DNA
restriction endonucleases
DNA ligases
restriction endonucleases aka
restriction enzymes
recognize and cleave DNA at specific sequences (recognition sequences or restriction sites)
cleave phosphodiester bond
methylases
catalyze methylation of host DNA to protect it from digestion by the host cell’s restriction endonucleases
restriction-modification system
the restriction endonuclease and the corresponding methylase
dna ligases
joins the DNA fragment to be cloned to a suitable cloning vector
types I and III restriction endonucleases
large, multisubunit complexes containing both endonuclease and methylase activities
type II restriction endonucleases
simpler than types I and III
require no ATP
catalyze the hydrolytic cleavage of DNA Phosphodiester bonds within the recognition sequence
restriction sequences for some type II endonucleases
normally cleave at a specific site
typically 4-6 bp long
palindromic
cleave same spot on each side
sticky ends
unpaired bases on the ends
-due to endonuclases making staggered cuts
-can base pair with each other or complementary sticky ends
blunt ends
no unpaired bases on the ends
-due to endonucleases making straight cuts
the DNA Segment to be cloned is generated by
PCR
PCR is used to add the restriction site
-including restriction endonuclease cleavage sites facilitates the subsequent cloning of amplified DNA
(2 different restriction enzymes used- to permit proper orientation of DNA insert to plasmid, prevents ligation)
cleavage of PCR-amplified DNA creates
sticky ends used to ligate the amplified DNA to a cloning vector
linkers
synthetic DNA fragments created to bridge ligated ends
multiple cloning site (MCS)
inserted DNA fragment with multiple recognition sequences for restriction endonucleases
-useful for inserting additional DNA at a later point
three popular cloning vectors:
- plasmids
- bacterial artificial chromosomes
- yeast artificial chromosomes
plasmid
circular DNA molecule that replicates separately from the host chromosome
usually have symbiotic role in cell
key features of E.coli plasmid:
- origin of replication (ori)= sequence where replication is initiated
- resistance genes
- recognition sequences for restriction endonucleases
transformation
laboratory process by which small plasmids are introduced into bacterial cells through heat shock treatment
-becomes less successful as plasmid size increases
electroporation
laboratory process by which small plasmids are introduced into bacterial cells through high-voltage pulses
-transiently renders the bacterial membrane permeable
selectable markers and screenable markers are used to
identify cells that take up plasmid DNA
selectable marker
either permits the growth of a cell (positive selection) or kills the cell (negative selection) under defined conditions
screenable marker
gene encoding a protein that causes the cell to produce a colored or fluorescent molecule (visual change)
bacterial artificial chromosomes
plasmid vectors developed to allow the cloning of very long segments of DNA
BACs= composed of the plasmid vector and large segments of cloned DNA
the BAC vector
-have stable origins of replications that maintain low copy numbers
-contain par genes from an F plasmid that direct the reliable distribution of the recombinant chromosomes at cell division
CamR
positive selection marker in the bac vector
lacZ
screenable marker in the bac vector
yeast artificial chromosomes
YACS= composed of the plasmid vector and large segments of cloned DNA
*ori + centromere + 2 telomeres + selectable markers X and Y
DNA cloned in a YAC can be altered to study
-the function of specialized sequences in chromosomes metabolism
-mechanisms of gene regulation and expression
shuttle vectors
plasmids that can be propagated in cells of 2+ species
-incorporate multiple replication origins or other elements
the YAC vector
contains elements to maintain a eukaryotic chromosome in the yeast nucleus:
-yeast origin of replication (ori)
-two selectable markers
-specialized sequences for stability and proper chromosome segregation at cell division
–centromere (CEN)
–two telomeres (TEL)
*for bacteria, large areas of DNA are hard to keep but in yeast, larger you get, the more stable you become
pulsed field gel electrophoresis
segregates genomic fragments following partial digestion with restriction endonucleases
-used to obtain a suitable fragment size
the stability of YAC clones
increases with the length of the cloned DNA segment (up to a point)
-inserts > 150,000 are very stable
-inserts < 100,000 bps are gradually lost during mitosis
YACS that lack a telomere at either end are
rapidly degraded
expressing a eukaryotic protein in a bacterium
eukaryotic genes have surrounding sequences needed for their transcription and regulation
-sequences do not function in bacteria
expression vectors
cloning vectors with transcription and translation signals needed for the regulated expression of a cloned gene
in principle, any organism can serve as a host to express proteins from a different species:
bacteria
yeast
insects and insect viruses
mammalian cells in culture
the most common hosts for protein expression
bacteria
advantages of using bacterial hosts
-regulatory sequences are well understood
-can express high levels of cloned proteins
-easy to store and grow
-efficient methods for transforming and extracting DNA
-can be grown in huge amounts
disadvantages of using bacterial hosts
-some heterologous proteins do not fold correctly
-proteins may not undergo necessary posttranslational modifications or proteolytic cleavage
-some gene sequences can be difficult to express
–many eukaryotic proteins aggregate into insoluble cellular precipitates (INCLUSION BODIES)
transcription from the lac promoter
-gene of interest fused to lactose operon promoter and regulatory sequences
-transcription occurs when lactose is added to the medium
-regulation is “leaky”
transcription from the bacteriophage T7 promoter and RNA polymerase
-cloned gene is fused to a T7 promoter and transcribed by T7 RNA polymerase
-affords tight regulation
principles underlying protein expression in yeast are the same as those for bacteria:
-cloned genes must be linked to appropriate promoters
-gene expression can be controlled by choosing an appropriate medium
advantages of using yeast hosts
-well-understood eukaryotic organism
-expression of eukaryotic genes can be more efficient
-proteins may be folded and modified more accurately
disadvantages of using yeast hosts
-heterologous proteins may not fold properly
-yeast may lack the enzymes needed to modify the proteins to their active forms
-certain features of the gene sequence may hinder expression of a protein
baculoviruses
insect viruses with double-stranded DNA genomes
-virus cannot make viral protein because no capsid
bacmids
large circular DNAs that include the entire baculovirus gene and sequences that allow replication of the bacmid in E.coli
-store genetic material for virus without making the virus
transfection
term used when the DNA used for transformation includes viral sequences and leads to viral replication
(recombinant bacmids are transfected into insect cells)
advantages of using bacmid systems
-wide range available commercially
-may successfully replicate protein-modification patterns and produce active, correctly modified proteins
disadvantages of using bacmid systems
not successful with all proteins
mammalian cells in culture
DNA introduced into mammalian cells using engineered mammalian viruses as vectors
advantages of mammalian cells in culture
-proteins can be expressed either transiently or permanently
-proper posttranslational modification can be ensured
disadvantages of mammalian cells in culture
super expensive
site-directed mutagenesis
technique used to individually replace specific amino acids
-alters the protein
-pcr and cleavage based
oligonucleotide-directed mutagenesis
technique used to create a specific DNA sequence change
-amplify ENTIRE plasmid, then degrade old DNA
deletions
performed by cutting out a segment with restriction endonucleases and ligating the remaining portion
fusion protein
product of a ligated gene containing parts of two different genes
tag
peptide or protein that binds a simple, stable ligand with high affinity and specificity
-fused to gene encoding target protein
-permits purification by affinity chromatography
use of tagged proteins in protein purification
-provides good yield and high purity
-may affect the properties of attached proteins
RT-PCR
reverse transcriptase PCR
uses reverse transcriptase to generate a DNA strand from an RNA template, followed by standard PCR protocols using DNA polymerase
qPCR
quantitative PCR or real time PCR is used to estimate relative copy numbers of particular sequences in a sample
AMOUNTS
DNA library
collection of DNA clones
specialized catalogs of genetic information
for
1. gene discovery
2. determination of gene or protein function
complementary DNAs (cDNAs)
double-stranded DNA fragments formed from mRNA templates
-relies on reverse transcriptase
-assume its from mRNA
cDNA library
population of clones created by inserting cDNA fragments into vectors and cloning
combinatorial gene libraries
library focusing on sequence variants within one gene
three levels of protein function
phenotypic function
cellular function
molecular function
phenotypic function
describes the effects of a protein on the entire organism
cellular function
describes the network of interactions a protein engages in at the cellular level
molecular function
describes the precise biochemical activity of a protein
transcriptome
the entire complement of transcribed RNAs present at a given moment in the cell
proteome
the entire complement of proteins present at a given moment in the cell
comparative genomics
process by which GENE FUNCTIONS can be assigned by using genome databases to perform genome comparisons
BLAST
genome annotation
converts the sequence of residues into useful information about the location and function of genes and other critical sequences
orthologs
genes that occur in different species but have a clear sequence and functional relationship to each other
paralogs
genes similarly related to each other within a single species
syteny
conserved gene order
-provides additional evidence for an orthologous relationship between genes at identical locations within the related segments
ex: human and mouse
RNA -seq
method that determines the RNAs that are transcribed from a genome under a given set of conditions
mass spectrometry
can accurately catalog and quantify the thousands of PROTEINS present in a typical cell
-complementary approach to RNA-seq
-provides information about how proteins are modified
green fluorescent protein (GFP)
jellyfish protein that serves as a useful location marker
-a target gene fused to the GFP gene generates a highly fluorescent fusion protein
-variants of other colors and characteristics also exist
IF
alternative approach for visualizing the endogenous protein that involves fixation (and death) of the cell
where the protein is
epitope tag
short protein sequence that is bound tightly by an antibody
knowing what a protein interacts with can suggest its function
the association of a protein of unknown function with one whose function is known can imply a functional relationship
techniques:
-purification of protein complexes (IP)
-yeast two-hybrid analysis
IP
process of precipitating a fusion protein (containing the gene of interest and a gene for an epitope tag) by antibodies to the epitope
-proteins that bind to the tagged protein will also precipitate
yeast two hybrid analysis
technique that relies on the properties of the Gal4 protein
-the two domains of Gal4pm must be brought together to function corectly
-probes molecular interactions in vivo
if you do not know binding partner *
CRISPR/CAS systems
how we make mutations now
mutating or deleting a gene provides a path to understanding a gene’s function
CRISPR/ Cas systems definition
specific for eukaryotic and mammalian- bacteria has a lot of other options so this is less common
“clustered, regularly interspaced short palindromic repeats”
CRISPR sequences
regularly spaced short repeats in the bacterial genome, surrounding sequences derived from phage pathogens that previously infected the bacterium
cas protein= nuclease
crispr sequences and cas protein are components of a
bacterial immune system
components of the crispr/cas complex:
guide RNAs= transcribed viral spaced sequences that are cleaved
trans-activating CRISPR RNA (tracrRNA)
1+ Cas proteins
the complex binds and destroys invading bacteriophage DNA by the Cas protein nuclease activities
transposons
segments of DNA that can move from one location to another in the genome
