Ch 10, 13, 14.1, 14.2 Flashcards by Kristen Mazowiecki

the collective techniques for obtaining, amplifying, and manipulating specific DNA fragments

DNA technologies

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the application of DNA technologies to specific biological, medical, or agricultural problems

Genetic engineering

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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

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The insulin gene is only expressed in ______ in the pancreas

beta cells

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beta cells do not produce enough insulin

type 1 diabetes

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beta cells unable to respond

type 2 diabetes

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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

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Translation of the spliced Ins mRNA produces a protein of ____ amino acids

110

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within a living organism

in vivo

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in a test tube or petri dish

in vitro

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method for DNA in vitro

Southern blot

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method for DNA in vivo

FISH - fluorescence in situ hybridization

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method for RNA in vitro

Northern blot

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method for RNA in vivo

In situ hybridization

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method for protein in vitro

Western blot

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method for protein in vivo

immunofluorescence

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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

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Blotting starts with _________ to separate molecules in a mixture based on their physical properties such as size and charge.

gel electrophoresis

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DNA and RNA migrate out of the wells toward the ________ charge

positive

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Gel electrophoresis can be carried out under ________________ conditions

non-denaturing or denaturing

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The last step in blotting is to use a ______ to visualize a specific molecule on the membrane.

probe

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to form a hybrid by performing a cross, to anneal complementary nucleic acid strands from different sources.

hybridization

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The use of X-ray film to detect radioactive materials, including nucleic acids.

autoradiography

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Cutting is carried out by __________________

bacterial restriction enzymes

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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)

1. ORI 2. Antibiotic resistant gene 3. 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

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