BIL 150 Chapter 20

PCR: cycle 3

2 of the 8 molecules match the target sequence and are the right length

process of using DNA microarray assays

A DNA microarray consists of tiny amounts of a large number of single stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array, or grid, of dots Ideally, these fragments represent all the genes of an organism The mRNAs from cells under study are reverse transcribed into cDNAs and a fluorescent label is added so the cDNAs can be used as probes on the microarray Different fluorescent labels are used for different cell samples so that mutliple samples can be tested in the same experiment The resulting pattern of colored dots reveals the dots to which each probe was bound and thus the genes that are expressed in teh cell samples being tested Microarray technology started taking off after several papers about it were published

process of gel electrophoresis

A gel made of a polymer as a molecular sieve to separate nucleic acids or proteins differing in size, electrical charge, or other physical properties as they move in an electric field Each sample, a mixture of different DNA molecules, is placed in a separate well near one end of a thin slab of agarose gel The gel is set in a small plastic support and immersed in an aqueous buffered solution in a tray with electrodes at each end The current is then turned on, causing the negatively charged DNA molecules to move toward the positive electrode Shorter molecules are slowed down less than longer ones, so they move faster through the gel After the current is turned off, a DNA binding dye is added that fluoresces in UV light The horizontal ladder of bands at the bottom of the gel is a set of restriction fragments of known sizes for comparison with samples of unknown length

SNP

A single base pair site where variation is found in at least 1% of the population is called a single nucleotide polymorphism SNP Once a SNP is identified that is found in all affected people, researchers focus on that region and sequence it In nearly all cases, the SNP itself does not contribute directly to the diseease in question by altering the encoding protein; in fact, most SNPs are in noncoding regions If the SNP and a disease associated allele are close enough to be genetically linked, scientists can take advantage of teh fact that crossing over between the marker and the gene is very unlikely during gamete formation Therefore, the marker and gene will almost always be inherited together, even though the marker is not part of the gene

process of stem cells maintaining their population and generating differentiated cells

A stem cell can divide into another stem cell and a progenitor cell (or into two stem cells or two progenitor cells) A progenitor cell can differentiate into one of several cell types, depending on external factors

in situ hybridization

A technique using nucleic acid hybridization with a labeled probe to detect the location of a specific mRNA in an intact organism.

CRISPR - Cas 9 system

Cas 9 is a bacterial protein that helps defend bacteria against bacteriophage infections in a system worked out by jennifer doudna and emmanuelle charpentier In bacterial cells, Cas9 acts together with a "guide RNA" made from the CRISPR region of the bacterial system Similar to the restriction enzyme described earlier, Cas9 is a nuclease that cuts double stranded DNA molecules However, while a given restriction enzyme recognizes only one particular DNA sequence, the Cas 9 protein will cut any sequence to which it is directed Cas9 takes its marching orders from a guide RNA molecule that it binds and uses as a homing device, cutting both strands of any DNA sequence that is exactly complementary to the guide RNA Scientists have been able to exploit the function of Cas9 by introducing a Cas9 guide RNA complex into a cell they wish to alter The guide RNA in the complex is engineered to be complementary to the "target gene" Cas9 cuts both strands of the target DNA and the resulting broken ends of DNA trigger a DNA repair system When there is no undamaged DNA for the enzymes of the repair system to use as a template, the repair enzymes rejoin the ends, sometimes introducing or removing nucleotides If the cut is directed to a coding portion of the gene, the rejoining process often alters the DNA sequence so that the gene no longer works properly

process of gene editing using the CRISPR Cas 9 system

Cas9 protein and guide RNA are allowed to bind to each other, forming a complex that is then introduced into a cell In the nucleus, the complementary sequence of the guide RNA binds to part of the target gene. The active sites of the Cas9 protein cut teh DNA on both strands The broken strands of DNA are "repaired" by the cell in one of two ways Scientists can disable "knock out" the target gene to study its normal function. No template is provided and repair enzymes insert and/or delete random nucleotides, making the gene nonfunctional OR If the target gene has a mutation, it can be repaired by providing a normal copy of the gene. Repair enzymes use the normal gene as a template and synthesize the correct gene sequence

cloning vector

DNA molecule that can carry foreign DNA into a host cell and be replicated there

PCR: extension

DNA polymerase adds nucleotides to the 3' end of each primer

where are corresponding mRNAs found in cloned individuals?

Detect the mRNA by nucleic acid hybridization with molecules of complementary sequence that we can follow in some way The complementary molecule, a short, single stranded nucleic acid that can be either RNA or DNA, is called a nucleic acid probe Using our cloned gene as a template, we can synthesize a probe complementary to the mRNA Each probe molecule is labeled during synthesis with a fluorescent tag so we can follow it Solution containing probe molecules is added to the cloned individual, allowing the probe to hybridize specifically with any complementary sequences on the many mRNAs in embryonic cells in which the gene is being transcribed Because this tenchique allows us to see the mRNA in place in the intact organism, this technique is called in situ hybriduation

nuclear transplantation

Differentiated cells from animals generally do not divide in culture, much less develop into the multiple cell types of a new organism Therefore, early researchers had to use a different approach to answer the question of whether differentiated animal cells are totipotent Their approach was to remove the nucleus of an egg (creating an enucleated egg) and replace it with the nucleus of a differentiated cell, a procedure called nuclear transplantaiton or somatic cell nuclear transfer If the nculeus from the differentiated donor cell retains its full genetic capability, then it should be able to direct development of the recipient celll into all the tissues and organs of an organism

two major potential uses for human iPS cells

First, cells from patients suffering from diseases have been reprogrammed to become iPS cells, which act as model cells for studying the disease and potential treatments Second, in the field of regenerative medicien, a patient's own cells could be reprogrammed into iPS cells, and then used to replace nonfunctional tissues

advantages of using RNA seq over microarrays

First, the procedure is not based on hybridization with a labeled probe, so it doesn't depend on having genomic sequences in hand Second, it can measure levels of expression over a very wide range, unlike microarrays, which cannot accurately measure either very low or very high levels Third, a careful analysis provides a wealth of information about expression of a particular gene

plant cloning in agriculture

For plants such as orchids, cloning is the only commerically practical means of reproducing plants In other cases, cloning has been used to reproduce a plant with valuable characteristics, such as resistance to plant pathogens

Robert briggs and Thomas king

Frog species Transplanted a nucleus from an embryonic or tadpole cell into an enucleated egg of the same species In gurdon's experiments, the transplanted nucleus was often able to support normal development of the egg into a tadpole However, he found that the potential of a transplanted nucleus to direct normal development was inversely related to the age of the donor: the older the donor nucleus, the lower the percentage of normal tadpoles Concluded that something in the nucleus does change as animal cells differentiate In frogs and most other animals, nuclear potential tends to be restricted more and more as embryonic development and cell differentiation progress

using restriction enzymes to make a recombinant DNA plasmid

Gene cloning and genetic engineering generally rely on the use of enzymes that cut DNA molecules at a limited number of specific locations These enzymes, called restriction endonucleases, or restriction enzymes, were discovered in the late 1960s by biologists doing basic research on bacteria Restriction enzymes protect the bacterial cell by cutting up foreign DNA from other organisms or phages Each restriction enzyme is very specific, recognizing a particular short DNA sequence, or restriction site, and cutting both DNA strands at precise points within this restriction site The DNA of a bacterial cell is protected from the cell's own restriction enzymes by the addition of methyl groups (-CH3) to adenines or cytosines within the sequences recognized by the enzymes Most restriction sites for enzymes are symmetrical, the sequence of nucleotides is the same on both strands when read in the 5'-3' direction The most commonly used restriction enzymes recognize sequences containing four to eight nucleotide pairs Because any sequence this short usually occurs (by chance) many times in a long DNA molecule, a restriction enzyme will make many cuts in such a DNA molecule, yielding a set of restriction fragments All copies of a given DNA molecule always yield the same set of restriction fragments when exposed to the same restriction enzymes The most useful restriction enzymes cleave the sugar phosphate backbones in DNA strands in a staggered manner The resulting double stranded restriction fragments have at least one single stranded end, called a sticky end To check the recombinant plasmids after they have been copied many times in host cells, a researcher might cut the products again using the same restriction enzyme, expecting two DNA fragments, one the size of the plasmid and one the size of the inserted DNA To separate and visualize the fragments, researchers carry out a technique called gel electrophoresis, which uses a gel made of a polymer as a molecular sieve to separate out a mixture of nucleic acid fragments by length

basic research and various applications of gene cloning

Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Human growth hormone treats stunted growth Protein dissolves blood clots in heart attack therapy

process of gene cloning

Gene inserted into plasmid (a cloning vector) Plasmid put into bacterial cell Host cell grown in culture to form a clone of cells containing the "cloned" gene of interest

GMO

Genetically modified organism made when DNA is removed from one organism and placed within the DNA of what can be a very different organism.

process of "next generation sequencing"

Genomic DNA is fragmented and fragments of 300 base pairs are selected Each fragment is isolated with a bead in a droplet of aqueous solution The fragment is copied over and over by a technique called PCR All the 5' ends of one strand are specifically "captured" by the bead Eventually, 10^6 identical copies of the same single strand, which will be used as a template strand, are attached to the bead The bead is placed into a small well along with DNA polymerases and primers that can hybridize to the 3' end of the single (template) strand The well is one of 2 million on a multiwell plate, each containing a different DNA fragment to be sequenced. A solution of one of four nucleotides required for DNA synthesis (deoxynucleoside triphosphate) is added to all wells and then washed off. This is done sequentially for all four nucleotides: dATP, dTTP, dGTP and then dCTP. The entire process is then repeated. In each well, if the next base on the template strand is complementary to the added nucleotide, the nucleotide is joined to the growing strand, releasing PPi, which causes a flash of light that is recorded The nucleotide is washed off and a different nucleotide is added. If the nucleotide is not complementary to the next template base, it is not joined to the strand and there is no flash The process of adding and washing off the four nucleotides is repeated until every fragment has a complete complementary strand. The pattern of flashes reveals the sequence of the original fragment in each well Sequencing by synthesis - sequenced nucleotides in parallel to machines, allows machines to identify in real time which of the four nucleotides is added

problems with bacterial expression systems

Getting a cloned eukaryotic gene to function in bacterial host cells can be difficult because certain aspects of gene expression are different in eukaryotes and bacteria To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector - a cloning vector that contains a highly active bacterial promoter just upstream of a restriction site where the eukaryotic gene can be inserted in the correct reading frame The bacterial host cell will recognize the promoter and proceed to express the foreign gene now linked to that promoter Another problem with expression cloned eukaryotic genes in bacteria is the presence of noncoding regions (introns) in most eukaryotic genes Introns can make a eukaryotic gene very long and unwieldy, and they prevent correct expression of the gene by bacteiral cells,which do not have RNA splicing machinery This problem can be surmounted by using a form of the gene that includes only the exons Complementary DNA or cDNA

cloning of Dolly

In 1997, researchers in scotland announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transfer from a differentiated mammary gland cell The researchers implanted early embryos into surrogate mothers Out of several hundred embryos, one successfully completed normal development and dolly was born, a genetic clone of the nucleus donor At the age of 6, dolly suffered complications from a lung infection often seen in sheep kept indoors and was euthanized Another cloned sheep from the experiment developed an unusual lung disease This led to speculation that this sheep's cells were in some way not as healthy as those of a normal sheep, possibly reflecting incomplete programming of the original transplanted nucleus Reprogramming involves epigenetic changes that lead to changes in chromatin structure

induced pluripotent stem cells iPS

In all these cases, researchers transformed the differentiated cells into a type of ES cell by using a retrovirus to introduce extra, cloned copies of four "stem cell" master regulatory genes The "deprogrammed" cells are known as induced pluripotent stem (iPS) cells because, in using this fairly simple laboratory technique to return them to their undifferentiated state, pluripotency has been restored

process of gene therapy

Insert RNA version of normal allele into retrovirus or other viral vector Let virus infect bone marrow cells that have been removed from the patient and cultured Viral DNA carrying the normal allele inserts into chromosome Inject engineered cells into patient

embryonic and adult stem cells

Many early animal embryos contain stem cells capable of giving rise to differentiated cells of any type Stem cells can be isolated from early embryos at a stage called the blastula stage or its human equivalent, the blastocyst stage In culture, these embryonic stem cells reproduce indefinitely, and depending on culture conditions, they can be made to differentiate into a wide variety of specizlied cells and sperm The adult body also has stem cells, which serve to replace nonreproducing specialized cells as needed In contrast to ES (embryonic stem) cells, adult stem cells are not able to give rise to all cell types in the organism, although they cna generate several defined types To the surprise of many, the adult brain has been found to contain stem cells that continue to produce certain kinds of nerve cells Researchres have also reported finding stem cells in skin, hair, eyes and dental pulp Although adunt animals have only tiny numbers of stem cells, scientists are learning to idenify and isolate these cells from various tissues and in some cases, to grow them in culture With the right culture conditions, cultured stem cells from adult amimals have been made to differentiate into various defined types of specialized cells, athough none are as versatile as ES cells Research with embryonic or adult stem cells is a source of valuable data about differentiation and has enormous potential for medical applicaitons The ultimate aim is to supply cells for the repair of damaged or diseased organs The developmental potential of adult stem cells is limited to certain tissues ES cells hold more promise than adult stem cells for most medical applications becasue ES cells are pluripotent, capable of differentiating into many different cell types When the main aim of cloning is to produce ES cells to treat disease, the process is called therapeutic cloning

Taq polymerase

Named after the bacterial species from which it was first isolated Thermus aquaticus Lives in hot springs Stability of its DNA polymerase at high temperatures is an evolutionary adaptation that enables the enzyme to function at tempteratures up to 95 degrees C

process of dideoxyribonucleotide chain termination sequence

One strand of a DNA fragment is used as a template for synthesis of a nested set of complementary fragments; these are further analyzed to yield the sequence Biochemist frederick sanger received the nobel prize in 1980 for developing this method

pros to using eukaryotic gene expression

RNA splicing many eukaryotic proteins will not function unless they are modified after translation

process of RT-PCR

RT - PCR begins by turning sample sets of mRNAs into double stranded DNAs with the corresponding sequences First, the enzyme reverse transcriptase is used to synthesize a complementary DNA copy of each mRNA in the sample, called a reverse transcript Poly A tail on the 3' end of mRNA allows for a short complementary strand of thymine to be added and used as a primer for synthesis of this DNA strand Following enzymatic degradation of the mRNA, a second DNA strand, complementary to the first, is synthesized by DNA polymerase The resulting double stranded DNA is called complementary DNA (cDNA) Lacks introns and can be used for protein expression in bacteria Next in RT PCR is the PCR step PCR is a way of rapidly making many copies of one specific stretch of double stranded DNA, using primers that hybridize to the opposite ends of the segment of interest When the products are analyzed on a gel, copies of the amplified region will be observed as bands only in samples that originally contained mRNA from the gene of interest An enhancement called quantitative RT-PCR (qRT - PCR) uses a fluorescent dye that fluoresces only when bound to a double stranded PCR product The newer quantitative PCR machines can detect teh light and measure the PCR product, thus avoiding the need for electrophoresis while also providing quantitative data, a distinct advantage RT PCR or qRT PCR can also be carried out with mRNAs collected from different tissues at one time to discover which tissue is producing a specific mRNA Process of RT PCR analysis of the expression of single genes cDNA synthesis is carried out by incubating the mRNAs with reverse transcriptase and other necessary components PCR amplification of the sample is performed using primers specific to the gene of interest Gel electrophoresis will reveal amplified DNa products only in samples that contained mRNA transcribed from the specific gene

why are bacterial plasmids widely used?

Readily obtained from commercial suppliers Manipulated to form recombinant plasmids by insertion of foreign DNA in a test tube (in vitro) Easily introduced into bacterial cells

process of creating a transgenic animal

Remove eggs froma female of the recipient species and fertilize them in vitro Meanwhile they have cloned the desired gene from the donor organism They then inject the cloned dna directly into the nuclei of the fertilized eggs Some of the cells integrate the foregin DNA, the transgene, into their genome and are able to express the foreign gene The engineered embryos that arise from these zygotes are then surgically implanted in a surrogate mother If the embryo develops successfully, teh result is a transgenic animal that expressed its new "foreign" gene

how restriction enzymes are used to clone a foreign DNA fragment into a bacterial plasmid

Restriction enzyme cuts the sugar phosphate backbones DNA fragment from another source is added. Base pairing of sticky ends produces various combinations DNA ligase seals the strands

process of making complementary DNA (cDNA) from eukaryotic genes

Reverse transcriptase is added to a test tube containing mRNA isolated from a sample of cells Reverse transcriptase makes the first DNA strand using the mRNA as a template and a short poly-dT as a DNA primer mRNA is degraded by another enzyme DNA polymerase synthesizes the second DNA strand, using a primer in the reaction mixture The result is cDNA, which carries the complete coding sequence of the gene but no introns

stem cells

Stem cells are relatively unspecialized cells that can both reproduce itself indefinitely and, under appropriate conditions, differentiate into specialized cells of one or more types Thus, stem cells are able to both replenish their own population and generate cells that travel down specific differentiation pathways

process of restriction enzymes and PCR in gene cloning

The PCR primers are designed so that the DNA fragments obtained by PCR have, at each end, a restriction site matching the one in the cloning vector Cut with same restriction enzyme used on cloning vector A gene that makes bacterial cells resistant to an antibiotic is present on the plasmid Recombinant DNa plasmids are added to bacterial host cells, which are then treated with antibiotic. Only cells that take up a plasmid will survive, due to the antibiotic resistant gene

how reliable is a genetic marker?

The greater the number of markers examined in a DNA sample, the more likely it is that the profile is unique to one individual The exact probability of a match depends on the frequency of markers in the general population Informatin on how common markers are in different ethnic groups is critical because these marker frequencies may vary considerably among ethnic groups and between a particular ethnic group and the population as a whole

cloning: single cell cultures

The successful cloning of whole plants from single differentiated cells was accomplished during the 1950s by F C steward and his students at Cornell university, who worked with carrot plants Found that differentiated cells taken from the root (the carrot) and incubated in culture medium could grow into normal adult plants, each genetically identical to teh parent plant These results showed that differentiation does not necessarily involve irreversible changes in the DNA In plants, mature cells can "dedifferentiate" and then give rise to all the specialized cell types of the organism Any cell with this potential is said to be totipotent

sticky ends

These short extensions can form hydrogen bonded base pairs with complementary sticky ends on any other DNA molecules cut with the same enzyme

RNAi

This experimental approach uses synthetic double stranded RNA molecules matching the sequence of a particular gene to trigger breakdown of teh gene's messenger RNa or to block its translation

summary of PCR

Three step cycle brings about a chain reaction that produces an exponentially growing population of identical DNA molecules During each cycle, the reaction mixture is heated to denature (separate) the strands of teh double stranded DNA, and then cooled to allow annealing (hydrogen bonding) of short, single stranded DNA primers complementary to sequences on opposite strands at each end of the target sequence; finally, a heat stable DNA polymerase extends the primers in the 5'-3- direction

gene cloning is used to ......

To make many copies of, or amplify, a particular gene and to produce a protein product from it A protein with medical uses can be harvested in large quantities from cultures of bacteria carrying a cloned gene for the protein

process of PCR

With PCR, any specific segment - the so called target species - in a DNA sample can be copied many times (amplified) within a test tube PCR requires double stranded DNA containing the target sequence, a heat resistant DNA polymerase, all four nucleotides and two 15-20 nucleotide single DNA strands that serve as primers One primer is complementary to one end of the target sequence on one strand; the second primer is complementary to the other end of the sequence on the other strand Three cycles Process of each cycle Denaturation: heat briefly to separate DNA strands Annealing: cool to allow primers to form hydrogen bonds with ends of target sequence Extension: DNA polymerase adds nucleotides to the 3' end of each primer Cycle 1 Yields 2 molecules Cycle 2 Yields 4 molecules Cycle 3 2 of the 8 molecules match the target sequence and are the right length

electroporation

a brief electrical pulse applied to a solution containing cells creates temporary holes in their plasma membranes, through which DNA can enter

expression vector

a cloning vector that contains a highly active bacterial promoter just upstream of a restriction site where the eukaryotic gene can be inserted in the correct reading frame

recombinant DNA molecule

a molecule containing DNA from two different sources, very different sources

nucleic acid hybridization

base pairing of one strand of a nucleic acid to a complementary sequence on a strnad from a different nucleic acid molecule

gene drive

biased inheritance of the engineered gene during reproduction rapidly "drives" the new allele through the population

PCR: annealing

cool to allow primers to form hydrogen bonds with ends of target sequence

genetic engineering

direct manipulation of genes for practical purposes

how to get DNA into a cell

electroporation injection by needle soil bacterium

gene profile

genetic markers

PCR: denaturation

heat briefly to separate DNA strands

process of using RNA sequences (RNA seq) to analyze expression of many genes

mRNAs are isoalted from the tissue being studied mRNAs are cut into similar sized, small fragments mRNAs are reverse transcribed into cDNAs of the same size cDNAs are sequenced The short sequences are mapped by computer onto the genome sequence. The resulting data, including the number of times a sequence is present, indicate which genes are expressed in a given tissue and at what level

biotechnology

manipulation of organisms or their components to make useful products

stem cell

relatively unspecialized cell that can both reproduce itself indefinitely and under appropriate conditions, differentiate into specialized cells of one or more types Stem cells ahve great potential for regenerating damaged tissues

plasmids

small circular DNA molecules that are replicated separately

gene therapy

the introdction of genes into an afflicted individual for therapeutic purposes - holds great potential for treating the relatively small number of disorders traceable to a single defective gene The aim of this approach is to insert a normal allele of the defective gene into the somatic cells of the tissue affected by teh disorder

methods for cloning DNA

use bacteria - most often E coli

PCR

used to obtain many copies of desired gene

why is PCR used?

used to provide the specific DNA fragment for cloning PCR primers are synthesized to include a restriction site at the end of the DNA fragment that matches the site in the cloning vector, and the fragment and vector are cut and ligated together The resulting plasmids are sequenced so that those with error free inserts can be selected

PCR: cycle 1

yields 2 molecules

PCR: cycle 2

yields 4 molecules