Ch 10, 13, 14.1, 14.2 Flashcards
the collective techniques for obtaining, amplifying, and manipulating specific DNA fragments
DNA technologies
the application of DNA technologies to specific biological, medical, or agricultural problems
Genetic engineering
the ultimate extension of the technologies to the global analysis of the nucleic acids present in a nucleus, a cell, an organism, or a group of related species
Genomics
The insulin gene is only expressed in ______ in the pancreas
beta cells
beta cells do not produce enough insulin
type 1 diabetes
beta cells unable to respond
type 2 diabetes
1.) the construction of an expression plasmid that contains a human insulin cDNA insert.
2.) The plasmid is then transformed into bacteria, and the bacteria are grown in large quantities.
3.) As the recombinant bacteria grow, they transcribe insulin mRNA and translate the mRNA into insulin protein.
4.) the bacteria are harvested, and the insulin protein is extracted and purified for therapeutic use in humans.
Production of human insulin by recombinant DNA technology
Translation of the spliced Ins mRNA produces a protein of ____ amino acids
110
within a living organism
in vivo
in a test tube or petri dish
in vitro
method for DNA in vitro
Southern blot
method for DNA in vivo
FISH - fluorescence in situ hybridization
method for RNA in vitro
Northern blot
method for RNA in vivo
In situ hybridization
method for protein in vitro
Western blot
method for protein in vivo
immunofluorescence
a commonly used in vitro method to detect and quantify a specific DNA, RNA, or protein molecule within a mixture of many different DNA, RNA, or protein molecules
blotting
Blotting starts with _________ to separate molecules in a mixture based on their physical properties such as size and charge.
gel electrophoresis
DNA and RNA migrate out of the wells toward the ________ charge
positive
Gel electrophoresis can be carried out under ________________ conditions
non-denaturing or denaturing
The last step in blotting is to use a ______ to visualize a specific molecule on the membrane.
probe
to form a hybrid by performing a cross, to anneal complementary nucleic acid strands from different sources.
hybridization
The use of X-ray film to detect radioactive materials, including nucleic acids.
autoradiography
Cutting is carried out by __________________
bacterial restriction enzymes
Cutting enzymes are endonucleases that cleave phosphodiester bonds between nucleotides at specific DNA sequences, called ___________
restriction sites
Restriction sites are _________, which means that both strands have the same nucleotide sequence but in antiparallel orientation
palindromic
A DNA fragment resulting from cutting DNA with a restriction enzyme.
restriction fragments
A map of the restriction sites in a piece of DNA.
restriction map
An in vitro method for amplifying a specific DNA segment that uses two primers that hybridize to opposite ends of the segment in opposite polarity and, over successive cycles, prime exponential replication of that segment only.
PCR (polymerase chain reaction)
The DNA template is denatured by ______, resulting in single-stranded DNA molecules
heat
__________ replicates the single-stranded DNA segments by extending from the annealed primers
Taq polymerase
one cycle of PCR consists of three main steps
denaturing, annealing, and extending
The polymerase chain reaction uses specially designed ______ to amplify specific regions of DNA in a test tube.
primers
A method to measure the amount of a specific DNA molecule in a sample.
quantitative PCR
DNA synthesized from a messenger RNA template through the action of the enzyme reverse transcriptase.
complementary DNA (cDNA)
cDNA is made from mRNA in vitro by a special enzyme called _____________
reverse transcriptase
cDNA synthesis begins with the ______ of mRNA from a tissue
purification
A method to amplify an RNA sequence.
reverse transcription-PCR (RT-PCR)
The creation of recombinant DNA molecules that can be replicated in cells.
DNA cloning
Any DNA to be used in cloning or in DNA-mediated transformation.
donor DNA (or insert DNA)
A DNA molecule generated in the laboratory that brings together pieces of DNA from multiple sources.
recombinant DNA
___________ are naturally occurring DNA molecules that serve as vehicles to carry foreign DNA into a cell
Cloning vectors
An enzyme involved in DNA replication and repair that seals the DNA backbone by catalyzing the formation of phosphodiester bonds.
DNA ligase
A region of a vector that contains multiple restriction sites that occur only once in the vector.
multiple cloning site (MCS) or polylinker
Because of sequence complementarity at the ends of the vector and insert, the vector and insert ______
anneal
Plasmid cloning vectors must be:
small in size; easy to work with; high copy number; accept insert of small size
An autonomously replicating extrachromosomal DNA molecule.
plasmids
Phage cloning vectors must:
accept insert up to 15 kb; more steps are needed to construct the clone; require packaging DNA into phage
What is the advantage of using BAC vector to construct a genomic library for large genomes?
The main advantage of using BACs for genomic library construction was the stability of the large, very low or single-copy (SC) clones
Essential features of cloning vectors: (3 things)
- ORI
- Antibiotic resistant gene
- Multiple restriction sites
Recombinant cells are what color in colonies?
White
Non-recombinant cells are what color in colonies?
Blue
Genomic library
A collection of clones that cover the entire genome of an organism
Clones are made using vectors that can accept large size inserts (>10kb)
Used for gene cloning, genome sequencing, mapping etc.
cDNA library
A collection of clones that represent mRNAs present in the sample
Clones are made using plasmid vectors
Used for gene cloning, expression analysis, isolate gene for making recombinant proteins etc.
PCR can’t be used to amplify:
An unknown gene from unsequenced organism
a method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication
Sanger sequencing
The dideoxynucleotides, or ddNTPSs, differ from the deoxynucleotides by:
the lack of a free 3′ OH group on the five-carbon sugar
What will happen if a ddNTP is incorporated into a new DNA strand?
If a ddNTP is added to a growing a DNA strand, the chain is not extended any further because the free 3′ OH group needed to add another nucleotide is not available
What is the reagent that results in chain termination in Sanger sequencing approach?
Dideoxynucleotides
Strategies to create site-specific DSBs:
Zinc-finger nucleases (ZFNs)
Transcription activator-like effector nucleases (TALENs)
CRISPR RNA-guided Cas nucleases (CRISPR-Cas). (clustered regularly interspaced short palindromic repeats - CRISPR-associated protein)
a specific, efficient and versatile gene-editing technology we can harness to modify, delete or correct precise regions of our DNA
CRISPR-Cas9 genome engineering
genes that determine the body plan, and the number, identity and pattern of body part.
Toolkit genes
one body part is replaced by another
homeotic transformation
_________ are expressed in spatially restricted domains and regulate the IDENTITY of body parts
Hox genes
Hox proteins are ________ factors
transcription
Homeodomain is a ____-binding domain
DNA
When scientists compare several protein sequences by sequence alignment, what are they trying to look for?
evolutionary relationships between genes and shared patterns among functionally or structurally related genes.
Bicoid mutants are missing the ______ region
anterior
a maternal effect gene that sets up the anteroposterior axis of the embryo
bicoid
The distinctive feature of maternal effect genes is that
the phenotype of the embryo depends solely on the genotype of the mother.
define continuous blocks of segments
GAP genes
define a pair of segments
Pair rule genes
affect patterning in each segment
Segment polarity genes
___________ repress mRNA translation to determine cell lineages
mRNA-binding proteins
A microRNA controls ___________________
developmental timing
the analysis of the information content of entire genomes, includes the numbers and types of genes and gene products as well as the location, number, and types of binding sites on DNA and RNA that allow functional products to be produced at the correct time and place.
Bioinformatics
considers the genomes of closely and distantly related species for evolutionary insight.
Comparative genomics
the use of an expanding variety of methods, including reverse genetics, to understand gene and protein function in biological processes.
Functional genomics
Whole-genome shot gun (WGS)
sequence first, map later
Order-clone sequencing
map first, sequence later
Sequencing reads are taken only of….
the ends of cloned inserts.
Repetitive elements found in many locations in the genome collapsed into a _________, creating a challenge for genome assembly.
single sequence contig
Traditional WGS
Sanger sequencing approach
Next generation WGS
Pyrosequencing (detects light emitted during the sequential addition of nucleotides during the synthesis of a complementary strand of DNA)
Advantages of Sanger
Longer sequence read, more accurate, easier to assemble
Advantages of pyrosequencing
Generates data faster, More cost-effective
Next-generation WGS sequencing differs from traditional sequencing in that next-gen WGS does not require
Overlapping sequence reads
______ architecture is the basis for human organ anatomy
Tubular
the formation of tubes (one of the fundamental morphogenetic events taking place during development)
Tubulogenesis
_______ factors direct salivary gland tubulogenesis
Nuclear
_____ is required for tube elongation and its function is tissue autonomous
Ribbon (rib)
Abnormal _______ in rib mutants
cell size and shape
Rib is expressed in….
all three germ layers
one of the most powerful tools for targeted gene expression. It is based on the properties of the yeast GAL4 transcription factor which activates transcription of its target genes by binding to UAS cis-regulatory sites.
GAL4/UAS system
combines chromatin immunoprecipitation with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins
ChIP sequencing
Rib binds ______
ribosomal protein genes
