Ch 4 DNA Technology

The order of nucleotides in a DNA strand or fragment is called

DNA sequence

DNA sequencing can be used for several purposes, including the determination of ________ and for the construction of _______

gene or genomic sequences; nucleic acid sequences for experimental purposes

The first widely used sequencing approach and employs dideoxy nucleotides (ddNTPs)

Sanger dideoxy method

ddNTPS can be incorporated into growing DNA strands via _______ involving the ddNTP _________

phosphodiester bonds; 5' phosphate groups

Incorporation of ddNTpS in this way results in the _______ of DNA strand elongation due to the lack of ________ to which new nucleotides can bind

termination; 3' hydroxyl groups

A technique for generating multiple copies of a DNA sequence

polymerase chain reaction (PCR)

The reagents required for PCR include the following:

- source DNA template

- primer pairs

- thermostable DNA polymerase

- deoxyribonucleoside triphosphates (dNTPs)

PCR occurs in a repeating cycle of three steps

Denaturation, Annealing, and Elongation

PCR in which product amplification is quantified in real time as the reaction progresses, typically by using fluorescent DNA markers

Real-time or quantitative PCR (qPCR)

Endonucleases that typically originate in bacteria and cut double-stranded DNA (dsDNA) at nonterminal sites (ie, at places other than dsDNA ends)

Restriction enzymes

In nature, restriction enzymes protect bacteria from _________ by cutting viral DNA into _______, thereby _______ transfer of foreign DNA into bacterial cells

bacterial viruses (ie, bacteriophages); small pieces; restricting

Some enzymes, like _____, cut evenly across dsDNA to create blunt ends or cuts in dsDNA that do not create single-stranded regions.

Alul

Other enzymes, like ______ cut unevenly across dsDNA, thereby creating short single-stranded segments known as sticky ends on each of the cut strands

EcoRI

The insertion of a previously isolated gene into a DNA sequence, typically to make multiple copies of the gene

Gene cloning

Gene cloning is often carried out for the ultimate purpose of creating a _____ amount of a specific gene's transcribed or translated products (______)

large; RNA or protein

The gene or gene products formed during cloning are called _____, meaning that they are from more than one source

recombinant

One approach to gene cloning consists of cutting ______ with a restriction enzyme and adding the _______ to be cloned along with a ______

plasmid DNA; DNA fragment; DNA ligase

Small, circular, nonchromosomal DNA, typically found in bacteria (may have a single origin of replication)

Plasmid DNA

The ends of the DNA fragment and the plasmid must ____ for it to be a successful incorporation of the fragment into the plasmid

match

A way to accomplish that is to match the ends of the DNA fragment and the plasmid is by

- using the same restriction enzyme in both the isolation of the DNA fragment and the cutting of the plasmid

- using PCR to add restriction sites at each end of the sequence to the cloned

When the use of different restriction enzymes produces a combination of noncomplementary blunt and sticky ends on the DNA fragments and the plasmid, _______ and _______ can be added to fill in the staggered ends. The resulting fragment and plasmid, both blunt-ended, are then joined by ______.

dNTPS and DNA polymerase; DNA ligase

After the addition of the DNA fragment, the _______ are added to bacterial cells, and the cells are ________ to take up the plasmids

plasmids; stimulated

To stimulate the cells, you would

heating up the cells

The process of taking up plasmids

transformation

An ______ gene is typically inserted into the plasmid with the DNA sequence containing the gene to be _______, and the bacterial colonies are then grown in media containing the antibiotic.

antibiotic resistance; cloned

DNA generated from an RNA template, typically mRNA

complementary DNA (cDNA)

Because _______ lacks introns, cDNA represents the _______ amount of genetic information necessary for gene expression: a contiguous sequence of nucleotides coding for a protein.

mature mRNA; minimum

To generate cDNA, _____ is first used as a template for the synthesis of a single cDNA strand

mRNA

The single ______ strand is then used as a template to synthesize a complementary second cDNA strand, thereby completing the synthesis of ______

cDNA; double-stranded cDNA

To initiate synthesis of the first cDNA strand, isolated mRNA is typically mixed with _______ composed of ______

primers ; thymine nucleotides

These primers, known as ______, are complementary to the ______of eukaryotic mRNA molecules

poly-T or oligo(dT) primers; 3' poly-A tails

The enzyme ______ uses the primer-bound mRNA as a template for the synthesis of DNA, adding ______ to generate single-stranded cDNA

reverse transcriptase; dNTPs

The mRNA strand is then partially ________ by the reverse transcriptase, leaving the first cDNA strand bound to the original RNA in some regions

degraded

The second (complementary) cDNA strand is then synthesized by ______, using the first cDNA strand as a template

DNA polymerase

The RNA fragments that remain bound to the first cDNA strand serve as _______ to which DNA polymerase successively adds _______

primers; dNTPs

Remaining RNA fragments are _______ during the synthesis of the second cDNA, as the _______ of DNA polymerase displaces NTPs as its adds dNTPs

removed; 5' --> 3' exonuclease activity

_______ is then added to seal any nicks in the DNA, completing the synthesis of the ______ cDNA

DNA ligase; double-stranded

Collections of cloned DNA fragments and can be generated in several ways

DNA libraries

The first approach to DNA library construction is to _______ (using restriction enzymes) or _______ (using sudden, forceful movements) break chromosomal DNA into smaller pieces of _______

enzymatically; mechananically; double-stranded DNA

A second approach for constructing a DNA library begins with mRNA, which is _______ into ______ before being inserted into ______ and transferred to recipient cells for amplification

reverse transcribed; double-stranded cDNA; plasmids

A third approach uses ______ to amplify cDNA of ______ or ______

PCR; individual genes; genomic fragments

Separating DNA molecules of different sizes by using an electric current to induce the molecules to migrate through a special gel

DNA electrophoresis

The bands produced during electrophoresis can be visualized using stains such as _____, which inserts between the nucleic acid bases and fluoresces under ultraviolet light

ethidium bromide

DNA bands can also be detected using a technique called

Southern blotting

Southern blotting uses a nucleic acid probe to detect only bands that contain a

specific DNA sequence

The base pairing of complementary strands of nucleic acids via hydrogen bonding

Hybridization

Are used to determine which part or parts of a sample contain the complementary nucleic acid sequence

Hybridization probes

The use of ionizing electromagnetic radiation for imaging or imaging-related measurements

Radiography

The type of radiography in which internally released ionizing radiation is used for imaging or measurement purposes

Autoradiography

RNA detection methods include hybridization to an ______, _____, and _____

RNA or DNA probe; autoradiography; UV absorption

The electrophoretic separation of RNA molecules followed by detection using a single-stranded nucleic acid (typically DNA) probe that hybridizes with a specific RNA sequence

Northern blotting

Consists of probes for many distinct sequences rather than a single sequence, also another method of detecting RNA

microarray

A complementary hybridization probe hybridizes with RNA molecules in a tissue sample, allows gene expression to be localized within a tissue or cell

In situ RNA hybridization

If the hybridization probe is labeled with a fluorescent tag, then fluorescence within the sample reveals the location(s) of complementary RNA molecules, and the method is called

fluorescence in situ hybridization (FISH)

Used to produce complementary DNA molecules from the RNA in a sample, and then PCR is used to amplify the cDNA

RT-PCR, reverse transcriptase

When the mathematical relationship between amplification cycle number and the amount of cDNA synthesized is used to quantify the amount of RNA in a sample, the process is referred to

RT-qPCR

A technique for measuring the amount of a specific protein in a sample using an immune-based detection mechanism

enzyme-linked immunosorbent assay (ELISA)

Proteins can also be studied using

polyacrylamide gel electrophoresis (PAGE)

Proteins separated on ______ retain their native structure and charge, both of which affect electrophoretic migration

nondenaturing (native) gels

Denaturing gels employ ______ such as _____ to impart a uniform charge to proteins

denaturants; sodium dodecyl sulfate (SDS)

This negative charge is _______ to size, minimizing charge differences between proteins, so separation of proteins by denaturing gel electrophoresis (eg, SDS-PAGE) occurs primarily on the basis of ______

proportional; size

Proteins in a sample can be electrophoretically separated, transferred to a membrane, and visualized in a process known as

western blotting

The protein bands are detected via binding to a primary antibody, to which a secondary antibody binds for visualization (eg, via chemiluminescence, fluorescence, or autoradiography

true

Prior to adding antibodies, membranes are typically exposed to _____ or another substance to block _____ binding sites, a step that reduces background binding

milk; nonspecific

Antibody-based detection of specific proteins can also be used for the visualization of proteins in histological sections

Immunohistochemistry

A means of studying protein-protein interactions using the affinity of a protein for a binding partner

Pulldown assays

Organisms or cells in which one or more genes have been removed or made nonfunctional

Knockout models

One approach for developing knockouts is derived from a bacterial defense system that collects ______ DNA segments from _____, enabling each bacterium to accumulate previously encountered viral DNA sequences

short; infecting viruses

The viral sequences are inserted at regular intervals between clusters of palindromic sequences of bacterial DNA, forming

clustered, regularly, interspaced, short, palindromic repeats (CRISPR)

In _______ gene editing, a synthetic sgRNA is employed to target a sequence of interest.

CRISPR-Cas 9

Another approach for generating knockouts, called the ______ strategy, employs a recombinase enzyme called Cre, which recognizes special DNA sequences called loxP sites

Cre-loxP

Cre cuts out the portion of the DNA between two ______ sites as well as ______ of the two loxP sites, then it ______ the two cut ends to leave a single loxP site

loxP; one; joins

A means of assessing the location of the gene(s) responsible for a recessively inherited phenotype

Complementation assay

Designed to post-transcriptionally modulate gene expression by manipulating mRNA function using short (-20-25 nucleotides), synthetic oligonucleotides that undergo Watson-Crick base pairing

Antisense strategies

Some of these oligonucleotides have counterparts in nature

RNA interference (RNAi)

Examples of RNAi

Short interfering RNA (siRNA) and mircoRNA (miRNA)

siRNA pairing is usually ______. This specificity makes the guide strand-target strand pair a target for ______ by a specific endonuclease, ultimately resulting in the enzymatic destruction of the target ______.

complete and specific; destruction; mRNA

miRNA undergoes ______ mRNA pairing, allowing miRNA to interact with ______ mRNA sequences rather than ______ specific mRNA sequence

partial; multiple; a single

miRNA typically either ______ translation of the bound mRNA or _____ of a pre-mRNA, although with extensive pairing mRNA ______ can be triggered

represses; alters splicing; destruction

Single-stranded synthetic antisense RNA molecules that hybridize with target mRNA and sterically block translation or alter splicing, similar to miRNA's role in RNAi.

Morpholinos

Morpholinos are ______ and possess a six-membered morpholine ring, which makes morpholinos ______ susceptible to degradation and is thought to ______ the likelihood of off-target effects

uncharged; less; reduce

An attempt to treat a condition or augment health by altering the complement of genes possessed or expressed

Gene therapy

The application of biotechnological methods or knowledge to questions about evidence in legal proceedings

forensics

A type of DNA analysis that focuses on differences in DNA fragments' lengths generated by the polymerase chain reaction or by treatment of DNA samples with an endonuclease

DNA fingerprinting

Well-adapted for survival in a specific type of harsh environment

extremophiles