Chapter 20

Early attempts at monitoring the course of PCR used what as a probe?

EtBr- Ethidium Bromide

Some vectors used in cloning experiments contain bacterial promoters that are adjacent to unique cloning sites. This makes it possible to insert a gene sequence next to the bacterial promoter and express the gene in bacterial cells. These are called expression vectors. If you wanted to express a eukaryotic protein in bacterial cells, would you clone genomic DNA or cDNA into the expression vector? Explain your choice.

It would be necessary to use cDNA so that the gene would not carry any introns. Bacterial cells do not contain spliceosomes (which are described in Chapter 12). To express a eukaryotic protein in bacteria, a researcher would clone cDNA into bacteria, because the cDNA does not contain introns.

Recombinant DNA molecules

Molecules that are produced in a test tube by covalently linking DNA fragments from two different sources

Where are the vectors commonly used in gene-cloning experiments originally derived from?

Plasmids

What is cDNA? In eukaryotes, how does cDNA differ from genomic DNA?

The term cDNA refers to DNA that is made using RNA as the starting material. Compared to genomic DNA, it lacks introns

PCR cycle

[[3 STEPS]] 1) denaturation 2) primer annealing 3) primer extension

The hydrogen bonding bw the sticky ends of DNA fragments promotes:

a temporary interaction bw the two fragments that is not completely stable bc only a few H-bonds are bw complementary bases

The function of reverse transcriptase is to:

copy RNA into DNA

Phosphodiester bond

covalently bonds the sugar-phosphate backbones of 2 different DNA strands after their complementary sticky ends have hydrogen bonded together -stabilizes the bond between the two fragments

What is used to cut DNA into pieces and join them together?

enzymes

What may be used to construct a DNA library?

genomic DNA or cDNA

What can cDNA be made from?

mRNA via reverse transcriptase

What does gene cloning involve?

the insertion of DNA fragments into vectors, which are then propagated within host cells

Fluorescence

- a thermocycler that has the capacity to measure changes in the level of fluorescence that is emitted by probes that are added to the PCR mixture can help [[determine the amount of a PCR product during real-time PCR]] - fluorescence given off by a probe depends on the amount of the PCR product

SYBR Green I

- cyanine dye that's frequently used in real-time PCR experiments instead of probes like TaqMan

Taq DNA polymerase

-one of the reagents needed for a PCR experiment -a thermostable form of DNA polymerase -necessary bc PCR involves heating steps that inactivate most other natural forms of DNA polymerase

Prior to the action of DNA ligase, how many hydrogen bonds are holding the two DNA fragments together?

8

What is a DNA library? Do you think this is an appropriate name?

A DNA library is a collection of recombinant vectors that contain different pieces of DNA from a source of chromosomal DNA. Because it is a diverse collection of many different DNA pieces, the name library seems appropriate.

What is a recombinant vector? How is a recombinant vector constructed? Explain how X-Gal is used in a method of identifying recombinant vectors that contain segments of chromosomal DNA.

A recombinant vector is a vector that has a piece of foreign DNA inserted into it. The foreign DNA came from somewhere else, such as the chromosomal DNA of some organism. To construct a recombinant vector, the vector and source of foreign DNA are digested with the same restriction enzyme. The complementary ends of the fragments are allowed to hydrogen bond to each other (i.e., sticky ends are allowed to bind), and then DNA ligase is added to create covalent phosphoester bonds. In some cases, a piece of the foreign DNA will become ligated to the vector, thereby creating a recombinant vector. In other cases, the two ends of the vector ligate back together, restoring the vector to its original structure. As described in Figure 20.2, the insertion of foreign DNA can be detected using X-Gal. As seen here, the insertion of the foreign DNA causes the inactivation of the lacZ gene. The lacZ gene encodes the enzyme β-galactosidase, which converts the colorless compound X-Gal to a blue compound. If the lacZ gene is inactivated by the insertion of foreign DNA, the enzyme will not be produced, and the bacterial colonies will be white. If the vector has simply recircularized, and the lacZ gene remains intact, the enzyme will be produced and the colonies will be blue.

What is a restriction enzyme? What structure does it recognize? What type of chemical bond does it cleave?

A restriction enzyme recognizes and binds to a specific DNA sequence and then cleaves a (covalent) phosphoester bond in each of two DNA strands

Real-time PCR

- allows a researcher to follow the amount of a specific PCR product in "real time" as PCR is taking place in a thermocycler - allows researchers to determine how much DNA, such as the DNA that encodes a specific gene, was originally in the sample before PCR was conducted - if the starting material is mRNA that is reverse-transcribed into DNA, real-time PCR can be used to determine how much mRNA from a specific gene was in a sample -provides a way to quantitatively measure gene expression

Reverse Transcriptase PCR

- can detect the expression of small amounts of RNA from a single cell -RNA is isolated from a sample and mixed w/ reverse transcriptase, a primer that binds near the 3' end of the RNA of interest, and dNTPs - this generates a single-stranded cDNA, which then can be used as a template DNA in a conventional PCR reaction [[END RESULT]]: the RNA has been amplified to produce many copies of DNA

What are the three steps involved in each cycle of PCR?

- denaturation - primer annealing - primer extension

Cycle threshold method

- during the exponential phase of PCR, the amount of PCR product is proportional to the amount of starting template that was initially added - exponential phase of PCR is analyzed to quantitate the initial template concentration via this method - cycle threshold is reached when the acclimation of fluorescence is significantly greater than the background level - phases of PCR cycle - real-time PCR at high, medium, and low concentrations of the starting template DNA - a comparison bw an unknown sample and standards of known concentrations

Primer annealing

- lower temperature - allows oligonucleotide primers to bind to template DNA

Thermal cycler

- machine that carries out PCR -automates the timing of each cycle -DNA sample, dNTPs, Taq polymerase, and an excess amount of primers are mixed together in a single tube - tube is placed in a thermocycler and machine is set to operate within a defined temp range and # of cycles - during each cycle: temp increases to denature the DNA strands and and then lowered to allow annealing and extension - typically each cycle lasts 2-3 min and involves 20-30 cycles of replication and takes a few hours to complete

Reporter molecule

- on one end of a TaqMan probe - emits fluorescence at a certain wavelength , but that fluorescence is largely absorbed by the nearby quencher -the close proximity of the reporter molecule to the quencher molecule prevents the detection of fluorescence from the reporter molecule - reporter is separated from quencher by the 5'-3' exonuclease activity of Taq polymerase cleaving the oligonucleotide in the TaqMan probe into individual nucleotides

Exponential increase

- region of interest is doubled w/ each cycle - [[2^n]] n=cycles to get total # of copies produced of region of interest

Quencher molecule

- the close proximity of the reporter molecule to the quencher molecule prevents the detection of fluorescence from the reporter molecule bc the quencher absorbs fluorescence - reporter is separated from quencher by the 5'-3' exonuclease activity of Taq polymerase cleaving the oligonucleotide in the TaqMan probe into individual nucleotides

Ethidium bromide

- used as a probe in early attempts at monitoring the course of PCR - fluorescent dye used to stain DNA -EtBr intercalates bw stacked bases in double-stranded DNA and becomes highly fluorescent when exposed to UV light -EtBr sheds water molecules: water is an efficient fluorescent quencher -tubes are measured at cycles 17, 21, 25, and 29 to determine fluorescence levels w/out removing any sample from the PCR tube - EtBr is more fluorescent when bound to double-stranded DNA

Polymerase Chain Reaction (PCR)

-A way to copy DNA without the aid of vectors and host cells -involves denaturation, primer annealing, and primer extension -method of amplifying a DNA region involving the sequential use of oligonucleotide primers and Taq polymerase -makes large amounts of DNA in a defined region that is flanked by 2 primers

Describe the important features of cloning vectors. Explain the purpose of selectable marker genes in cloning experiments.

-All vectors have the ability to replicate when introduced into a living cell. -This ability is due to a DNA sequence known as an origin of replication, which determines the host cell specificity of a vector. -Modern vectors also contain convenient restriction sites where geneticists can insert fragments of DNA. -These vectors also contain selectable markers, which are genes that confer some selectable advantage for the host cell that carries them. -The most common selectable markers are antibiotic-resistance genes, which confer resistance to antibiotics that would normally inhibit the growth of the host cell.

Hydrogen bonding

-Bonds the ends of two different DNA pieces together bc of their complementary sticky ends -promotes interaction bw two DNA fragments

Origin of replication

-DNA sequence contained in a plasmid -recognized by replication enzymes of the host cell and allow it to be replicated -sequence determines whether or not the vector can replicate in a particular host cell

Discuss three important advances that have resulted from gene cloning:

-First, cloned genes can be used for DNA sequencing. This has allowed researchers to understand genetics at the molecular level -Second, cloned genes can be mutated using site-directed mutagenesis to identify gene sequences such as promoters and regulatory elements and to see how specific mutations alter the structure and function of DNA -Third, cloned genes can be used as probes to identify similar genes or RNA. Fourth, cloned genes can be introduced into a different cell type of species

In your own words, describe the series of steps necessary to clone a gene. Your answer should include the use of a probe to identify a bacterial colony that contains the cloned gene of interest.

-First, the chromosomal DNA that contains the source of the gene that you want to clone must be obtained from a cell (tissue) sample. A vector must also be obtained. -The vector and chromosomal DNA are digested with a restriction enzyme. They are mixed together to allow the sticky ends of the DNA fragments to bind to each other, and DNA ligase is then added to promote covalent bonds, hopefully creating a recombinant vector. -The DNA is introduced into a living cell. The vector will replicate and the cells will divide to produce a colony of cells that contain cloned DNA pieces. -To identify colonies that contain the gene you wish to clone, you must use a probe that will specifically identify a colony containing the correct recombinant vector. -A probe may be a DNA probe that is complementary to the gene you want to clone or it could be an antibody that recognizes the protein that is encoded by the gene.

How does gene cloning produce many copies of a gene?

-In conventional gene cloning, many copies are made because the vector replicates to a high copy number within the cell, and the cells divide to produce many more cells. -In PCR, the replication of the DNA to produce many copies is facilitated by primers, deoxyribonucleoside triphosphates (dNTPs), and Taq polymerase

What is the functional significance of sticky ends in a cloning experiment? What type of bonding makes the ends sticky?

-Sticky ends, which are complementary in their DNA sequence, promote the binding of DNA fragments to each other. -This binding is due to hydrogen bonding between the sticky ends.

Describe how cDNA is made

-[[RNA AS STARTING MATERIAL]] -short strand of DNA (oligonucleotide) that's a primer composed of thymine-containing nucleotides -mixed with mRNAs in the beginning of a cloning experiment -mRNA naturally contain a polyA tail at the 3' end -poly-dT primers are complimentary to the 3' end of mRNA -reverse transcriptase and dNTPs are added to make a DNA strand completely complementary to the mRNA -RNaseH partially digests the RNA generating short RNAs that are used as primers by DNA polymerase I to make a second DNA strand that is complementary to the strand made by reverse transcriptase -DNA ligase seals any nicks in this second DNA strand

Host cell

-a cell that harbors a vector -when a vector is replicated within a host cell, the DNA that it carries is also replicated

What specific function does the host cell play in gene/DNA cloning?

-able to replicate a vector and the DNA contained inside the vector

Primer extension

-after primer annealing, temp is raised slightly - Taq polymerase catalyzes the synthesis of complementary DNA strands in the 5'-3' direction starting at the primers - doubles the amount of the template DNA

Blue/white screening

-allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. -DNA of interest is ligated into a vector. -The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. -Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids (i.e. only the vector) grow into blue colonies. -This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used.

What are the functions of the selectable marker: lacZ, X-gal, and IPTG in blue/white screening?

-bacteria grown in the presence of X-gal and IPTG (an inducer of the lacZ gene) form blue colonies if they have a functional lacZ gene and contain recircularized vectors -if white colonies are formed then they do not have a functional lacZ gene contain recombinant vectors

Why can a bacteria containing the ampR gene grow on a media containing ampicillin?

-because they can degrade it -if the ampR gene is found within the plasmid, the growth of cells in the presence of ampicillin identifies bacteria that contain the plasmid

Competent cells

-cells that can take up DNA from the extracellular medium

Sticky ends

-certain types of restriction enzymes are useful in cloning bc they digest DNA into fragments with "sticky ends" *single-stranded regions of DNA that can hydrogen bond to a complementary sequence of DNA from a different source*

X gal

-colorless compound -used as a visual indication of whether a cell expresses a functional β-galactosidase enzyme -recircularized vector without an insert; B-galactosidase is made and converts x-gal BLUE -recombinant vector with an insert;B-galactosidase is inactive bc of the inserted DNA

Antibiotic resistance

-component of selectable markers -allows host cells to grow in presence of toxic substance

cDNA library

-contains recombinant vectors with cDNA inserts 1-cDNA made via transcriptase 2-to insert DNA into small vectors, short oligonucleotides (poly-dT primers) are attached to cDNAs via DNA ligase 3-poly-dT primers (linkers) contain DNA sequences w/ a unique site for a restriction enzyme 4-after linkers attach to cDNA, the cDNAs and the vectors are cut with restriction enzymes and then ligated to each other: produces cDNA library

The restriction enzymes used in gene-cloning experiments------, which generates sticky ends that can------

-cut the DNA -hydrogen bond w/ complementary sticky ends

Cloning

-enables geneticists to probe relationships between gene sequences and phenotypic consequences -many copies of a DNA fragment that are propagated within a vector or produced by PCR.

lacZ gene

-encodes beta-galactosidase -chromosomal DNA is inserted into a region of the vector that contains lacZ gene and disrupts the lacZ so it's no longer able to produce a functional enzyme

Amp^R

-encodes enzyme beta-lactamase: degrades ampicillin, an antibiotic that normally kills bacteria

Selectable marker

-gene -contain resistance genes which provide the host cells with the ability to grow in the presence of a toxic substance -the expression of the gene selects for the growth of the host cells

Denaturation

-in order to make copies of the DNA, template DNA is first denatured by heat treatment causing the strands to separate with high temp

IPTG

-inducer of the lacZ gene -nonmetabolizable lactose analog that can induce the lac promoter -bacteria grown in the presence of x-gal and IPTG form blue colonies if they have a functional lacZ gene and white colonies if they do not

What is a palindromic sequence and how is it relevant to the function of some restriction enzymes?

-most restriction enzymes cut at palindromic sequences -restriction enzymes bind to the DNA only in one specific configuration and are able to recognize palindromic sequences -Recognising a palindromic sequence enables them to cut both strands of DNA at the "same" site, because the strand will have the same sequence only in different directions at that site

Primers

-oligonucleotide -short segments of DNA, about 15-20 nucleotides in length -2 primers bind to specific sites in the DNA bc their bases are complementary these sites -[[END RESULT]]: the region that is flanked by the primers, which contained the gene of interest, is amplified, many copies of the region have been made and the region bw the 2 primers has been cloned

dNTPs

-one of the reagents needed for a PCR experiment -also called primers or oligonucleotides

Template DNA

-one of the reagents needed for a PCR experiment -sample of DNA that contains a gene of interest -reverse primer binds on the 3' end on the template DNA

What may be used as a vector in a gene cloning experiment?

-plasmid -virus

TaqMan Probe

-probe used in accordance with fluorescence that's added to a PCR mixture -oligonucleotide that has a reporter molecule at one end and a quencher molecule at the other - complementary to a site within the PCR product of interest - a primer and a TaqMan probe both anneal to the template DNA during primer annealing step

Palindromic sequence

-recognized by restriction enzymes -the sequence in one strand is identical when read in the opposite direction in the complementary strand

Restriction enzymes

-restriction endonuclease -cuts DNA into fragments that will be able to be insert chromosomal DNA into a plasmid or viral vector -bind to a specific base sequence and then cleave the DNA back-bone at two defined locations, one in each strand

Poly-dT primer

-short strand of DNA (oligonucleotide) that's a primer composed of thymine-containing nucleotides -mixed with mRNAs in the beginning of a cloning experiment -mRNA naturally contain a polyA tail at the 3' end -poly-dT primers are complimentary to the 3' end of mRNA -[[NEXT STEP]]: reverse transcriptase and dNTPs are added to make a DNA strand completely complementary to the mRNA

Plasmids

-small circular pieces of DNA -a natural source in many strains of bacteria and carry genes that confer resistance to antibiotics or other toxic substances

Vector

-small segment of DNA -a small DNA molecule that can replicate independently of host cell chromosomal DNA and produce many identical copies of an inserted gene

DNA ligase

-stabilizes the bond bw the sticky ends of two different DNA fragments and makes the bond more permanent -catalyzes the covalent bond formation (or linkage) in the sugar-phosphate backbones of both DNA strands after the sticky ends have hydrogen bonded with each other

Transformation

-step in gene cloning procedure where competent cells take up DNA from the extracellular medium

What is an advantage of making a cDNA library rather than a genomic library?

-the advantage of a DNA library is that it lacks introns -useful bc if a researcher wants to focus their attention on the coding sequence of a gene or if they want to express the gene in cells that so not splice out introns

Recombinant vector

-the vector containing a piece of chromosomal DNA -a fragment of DNA is ligated to both ends of the vector; a segment of chromosomal DNA has been inserted into the vector

Reverse transcriptase

-uses RNA as a template to make a complementary strand of DNA -encoded in the genome of retroviruses -provides a way for for retroviruses to copy their RNA genome into DNA molecules that integrate into the host cell's chromosome

cDNA

-when DNA is made from RNA as the starting material, the DNA is called complementary DNA -can be single or double stranded

Genomic library

-when the starting material is chromosomal DNA -plasmid or viral vectors that'll produce viral plaques or bacterial colonies

What is the order of steps in a gene-cloning experiment involving vectors?

1) Incubate the chromosomal DNA and the vector DNA w/ a restriction enzyme 2) Mix the chromosomal DNA and vector DNA together 3) Add DNA ligase 4) Introduce the DNA into living cells

Cloning experiments may involve two kinds of DNA molecules:

1) chromosomal DNA 2) vector DNA

What is the procedure for cloning a gene into a vector?

1) chromosomal DNA is isolated and digested with a restriction enzyme 2) enzyme cuts the chromosomes into many small fragments 3) plasmid DNA is also cut w/ same restriction enzyme at only one unique site 4) plasmid now has 2 ends complementary to the sticky ends of the chromosomal DNA fragments 5) the digested chromosomal DNA and plasmid DNA are mixed together and incubated under conditions that promote the binding of these complementary sticky ends 6) DNA ligase is added to catalyze the covalent linkage bw DNA fragments 7) - *recircularized vector* will ligate back together the two end of the vector - *recombinant vector* a fragment of chromosomal DNA is ligated to both ends of the vector and a segment of chromosomal DNA has been inserted into the vector 8) after ligation, begins the *transformation* the DNA is introduced into living cells treated w/ agents that render them permeable to DNA molecules *competent cells* 9) [[TO DETERMINE WHICH CELLS HAVE TAKEN UP A PLASMID]]: introduce plasmid to bacterial cells originally sensitive to ampicillin 10) bacteria is streaked onto plates containing bacterial growth media and ampicillin -a bacterium that has taken up a plasmid containing the ampR gene continues to divide and forms a bacterial colony of tens of millions cells all derived from the same original cell and the same type of plasmid DNA 11) [[TO DETERMINE WHICH BACTERIAL COLONY HAS RECIRCULARIZED VECTORS VS RECOMBINANT VECTORS]]: chromosomal DNA is inserted into a region of vector that contains the lacZ gene which encode B-galactosidase 12) insertion of chromosomal DNA into the vector disrupts lacZ gene so its not able to produce a functional enzyme *recircularized vector has a functional lacZ gene* 13) functionality of lacZ gene is determined by adding x-gal into the growth medium 14) x-gal is cleaved by B-galactosidase into a blue dye 15) bacteria grown in the presence of x-gal and IPTG(lacZ gene inducer) form blue colonies is they have a functional lacZ gene and white colonies if they do not 16) *bacterial colonies containing recircularized vectors form blue colonies, colonies containing recombinant vectors are white*

How do the ends of two different DNA pieces bond to each other?

The ends of two different DNA pieces hydrogen bond to each other bc of their complementary sticky ends

Compare and contrast a genomic library vs a cDNA library

[[GENOMIC LIBRARY]] - includes all possible fragments of DNA from a given cell or organism -larger -represents entire genome of an organism having both coding and noncoding regions -expression of genes taken from genomic library is difficult in prokaryotic systems like bacteria bc of absence of splicing mechanism -vectors used genomic library include plasmid in order to accommodate large fragments [[cDNA LIBRARY]] -only carries expressed gene sequences -smaller -represents only the expressed part of the genome and contain only coding sequences called ESTs -has only one coding sequence and can only be directly expressed in prokaryotic systems -vectors used include plasmids to accommodate small fragments bc cDNA has no introns

A collection of recombinant vectors that contain fragments of chromosomal DNA is called:

a genomic library

Gene cloning

the production of many copies of a gene using molecular methods, such as PCR or the introduction of a gene into a vector that replicates in a host cell

Explain the role of the selectable marker gene in a gene cloning experiment:

the selectable marker gene selects for the growth of bacteria that have taken up a plasmid

Explain the meaning of the term reverse transcriptase

this enzyme catalyzes the opposite of transcription and uses an RNA template to make DNA, whereas during transcription a DNA template is used to make RNA

What is the purpose of vector DNA?

to act as a carrier of the DNA segment that is to be cloned

What is Reverse transcriptase PCR used for?

to amplify RNA

What is real-time PCR used for?

to quantitate the amount of a specific gene or mRNA in a sample