Chapter 7 Molecular Biology Of The Gene Watson

1. How does the DNA migrate through the gel when an electrical field is applied for electrophoresis? Explain your choice and DNA is visualized after electrophoresis?

1. During gel electrophoresis the DNA migrates towards the positive electrode side because the DNA is negatively charged, Because of the negatively charged phosphate backbone present in DNA. During gel electrophoresis the DNA molecules are labeled with a fluorescent dye. The stain allows us to see patterns within the gel electrophoresis.

The most common host used to propagate DNA is the bacterium E. coli. Vector DNAs typically have three characteristics. What are they?

1. They contain an origin of replication that allows them to replicate inde- pendently of the chromosome of the host. (Note that some yeast vectors also require a centromere.) 2. They contain a selectable marker that allows cells that contain the vector (and any attached DNA) to be readily identified. 3. They have unique sites for one or more restriction enzymes. This allows DNA fragments to be inserted at a defined point within the vector such that the insertion does not interfere with the first two functions.

Two methods for labeling DNA

1. adding a label at the end of an intact DNA molecule. (phosphate radioactive therefore the DNA molecule is labeled. ) 2. Labeling by incorporation or synthesizing DNA in the presence of a labeled precursor.( usually nucleotides modified with fluorescent moiety or radioactive atoms ( Use PCR for this synthesis)

12. You want to characterize the developmental expression of the gene in Drosophila melanogaster. You isolate mRNA from embryos and adult flies and perform a northern blot using a labeled DNA probe specific to Gene Z mRNA, a gene required for development. The results are depicted below. Northern blot Adult embryo (male) of the cDNA of Protein Z Western blot Adult embryo (male) terminus of Protein Z Intrigued, you isolate protein Z from embryos and adult flies and perform a western blot using an antibody against the carboxyl terminus of the protein. The results are depicted below. You are surprised to find a single band of the same molecular weight in both embryos and adult flies. A. Propose a hypothesis to explain these results. B. Propose a modification to the western blot experimental strat- egy that would allow you to test your hypothesis. Assume you have access to any necessary reagents.

12. A. Looking at the tables I see that the western blot uncovered one band for Protein Z in the embryo and adult lanes where on the other hand the northern blot shows that there are two transcripts for Gene Z in embryonic flies but only one transcript in adult flies. My hypothesis is would state that antibody used in the western blot does not recognize the form of the protein as in the northern blot. B. In order to better my hypothesis I would test this by using new antibody with Protein Z in the western blot. If I added the new antibodies and saw a second band on the western blot in the embryo lane the data would support the hypothesis because the antibodies are what was not recognized.

3. Generally describe two methods for labeling a DNA probe

3. The two methods for labeling a DNA probe are labeling by incorporation and end labeling. These two methods are used to tag a DNA probe. The end labeling technique is done by labeling a DNA probe by using a g-phosphate from ATP and adding it to the 5' end of the OH on the DNA probe. The other method (incorporation) requires the DNA probe to be synthesized by the labeled nucleotides through PCR.

32P or 35S do what?

32P or 35S are usually the labeled precursors that flources at certain uv light wavelengths. The 32P or 35S is incorporated into the alph-phosphate pf one of the 4 nucleotides. The phtomultipers( light) excites the molecules and the 32P or 35S emits beta particulars. (pg. 151)

7. The following times and temperatures are an exam- ple of the steps for PCR. You can use Figure 7-12 to help you answer the following questions. 94°C => 94°C => 55°C => 72°C => 72°C =» 4°C 10 min ^ 30 sec 30 sec 1:30 sec ^ 10 min " (x 25 cycles) A. Why is the first step is carried out at 94°C? B. What happens in the reaction when the temperature shifts to 55°C during cycling? C. During cycling, what occurs when the temperature is at 72°C?

7. A. The high temperature disrupts the hydrogen bonding of the double-stranded DNA, which allows the strands denature. B. The primers anneal to the single-stranded regions of the DNA also known as re-annealing. C. The DNA polymerase carries out DNA synthesis by extending the annealed primers when back at 72 C.

How does a shotgun library work?

A "shotgun" library con- taining random genomic DNA inserts of 5 kb in length. Each well on the plate con- tains a different insert. Sequences 600 bp in length are determined for both ends of each genomic DNA (color coded). These paired-end sequences are used to align dif- ferent contigs. In this example, the 5-kb genomic DNA fragment with the blue se- quences contains matching sequences with contig A and contig B.

DNA Library

A DNA library is a population of identical vectors that each contains a differ- ent DNA insert (Fig. 7-9). To construct a DNA library, the target DNA (e.g., human genomic DNA) is digested with a restriction enzyme that gives a desired average insert size, ranging from <100 bp to more than a megabase (for very large insert sizes, the DNA is typically incompletely cut with a restriction enzyme). The cleaved DNA is then mixed with the appropriate vector cut with the same restriction enzyme in the presence of ligase. This creates a large collection of vectors with different DNA inserts.

What is a southern blot hybridization?

A Southern blot. DNA fragments, generated by digestion of a DNA molecule by a restriction enzyme, are run out on an agarose gel. Once stained, a pattern of fragments is seen. When transferred to a filter and probed with a DNA fragment homologous to just one sequence in the digested molecule, a single band is seen, corresponding to the position on the gel of the fragment containing that sequence. This was discovered by Edward Southern in order to identify a particular fragments contain the gene of interest.The technique of Southern blot hybridiza- tion (named after its inventor Edward Southern) will identify within the smear the size of the particular fragment containing the gene of interest.

6.How is genomic DNA library different from cDNA library? What is an advantage of using a cDNA library?

A genomic library consists of the complete set of DNA fragments, generated by restriction endonuclease digestion of the entire genome. A cDNA library, which consists only of expressed sequences in genomic DNA, is generated by reverse transcription of all mRNA in the cell. In each case, the resulting fragments of DNA are ligated into plasmid vectors. The human genome includes a large proportion of non-coding DNA includ- ing sequences that code for introns that are spliced out of the mRNA. cDNA libraries are useful for studying and expressing these gene-encoding sequences.

Gel Martix

A jelly like material that DNA Aand RNA flows through during Gel Electr.

Paired-end sequencing

A major limitation to producing larger contigs is the occurrence of repetitive DNAs (see Chapter 8). Such sequences complicate the assembly process because random DNA fragments from unlinked regions of a chromosome or genome might appear to overlap because of the presence of the same repetitive DNA sequence. One method that is used to overcome this diffi- culty is called paired-end sequencing. This is a simple technique that has produced powerful results (see Fig. 7-19).

Northern Blot

A similar procedure called northern blot hybridization (to distinguish it from Southern blot hybridization) can be used to identify a particular mRNA in a population of RNAs. Because mRNAs are relatively short (typically <5 kb), there is no need for them to be digested with any enzymes (there are only a limited number of specific RNA-cleaving enzymes). Otherwise, the proto- col is similar to that described for Southern blotting. The separated mRNAs are transferred to a positively charged membrane and probed with a probe DNA of choice. (In this case, hybrids are formed by base pairing between complementary strands of RNA and DNA.)

2. The restriction endonuclease, Xhol, recognizes the sequence 5'-CTCGAG-3' and cleaves between the C and T on each strand. A. What is the calculated frequency of this sequence occurring in a genome? B. The restriction endonuclease, Sail, recognizes the sequence 5'-GTCGAC-3' and cleaves between the G and T on each strand. Do you think the sticky ends produced after Xhol and Sail cleavage could adhere to each other? Explain your choice.

A. For a restriction enzyme that recognizes a 6-bp sequence, the frequency of finding that sequence in a given genome is 1 in 46 or 1 in 4096 bp. B. Yes. Even though the recognition sequences differ for XhoI and SalI, the sticky ends can base-pair with each other because the single-stranded regions are complementary to each other.

10. Three assays for testing interactions between protein and DNA are the electromobility shift assay (EMSA), DNA footprinting, and chromatin immunoprecipitation (ChIP). A. In a DNA footprinting assay, explain why only one strand of the DNA can be end-labeled for the experiment to work. B. Following the immunoprecipitation step in a chromatin immunoprecipitation (ChIP), explain how to identify the DNA sequences that remain bound to the protein of interest

A. Only one end (strand) of the DNA is labeled so that nuclease digestion of the bound DNA fragment will produce, after gel electrophoresis, a visible ladder of fragments extending from a single labeled end. Digestion of a strand labeled at both ends would complicate the pattern and obscure the "footprint." And, if the protein binds asymmetrically, the pattern becomes even more complicated. B. To determine if a specific known region is bound to the pro- tein, use primers that are specific to those sequences to amplify that sequence and compare the results to necessary controls. Another option is to use a tiling DNA microarray to identify many different sequences.

11.You decide to perform dideoxy sequencing on a PCR product. You add the appropriate 32 P-labeled primer, DNA polymerase, DNA template (the PCR product), buffer, dNTP mix, and a small amount of one of the four ddNTPs to four reaction tubes. You run the reactions in the thermal cycler, load each reaction into a separate lane of a polyacryla- mide gel, and separate the products by gel electrophoresis. In the figure below, the lanes are labeled according to the ddNTP added. ddATP ddTTP ddCTP ddGTP C D A. What is the sequence of the template strand? Be sure to label the 5' and 3' ends. B. Suppose that you accidentally added 10-fold more ddGTP to the reaction in lane 4. What effect would that have on the banding pattern in lane 4? C. In lane 5, draw what you would expect to see if you prepared a reaction using a nucleotide mix containing only dATP, dTTP, dCTP, and dGTP. D. Inlane6, draw what you would expect to see ifyou prepared a reaction using a nucleotide mix containing only ddATP, dTTP, dCTP, dGTP.

A. The templates strand would be 5' CATTGAGACCGT 3' B. If there was an excess amount of ddGTP in the reaction would increase the chance of stopping the DNA synthesis at Gs. If that happened we would see thicker bands at the bottom of the ddGTP lane and a lighter band at the top of the lane. C/ D. Answer below Lane 5 & 6

Fusion proteins

Alternatively, purified DNA sequences can be recombined to generate DNAs that encode so-called fusion proteins — that is, hybrid proteins made up of parts derived from different proteins.

Transformants

An antibiotic to which the plas- mid imparts resistance is then included in the growth medium to select for the growth of cells that have taken up the plasmid DNA — these cells are called transform ants. In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s)

Why Northern Blot?

An investigator might perform northern blot hybridization to ascertain the amount of a particular mRNA present in a sample rather than its size. This measure is a reflection of the level of expression of the gene that encodes that mRNA. Thus, for example, one might use northern blot hybridization to ask how much more mRNA of a specific type is present in a cell treated with an inducer of the gene in question compared with an uninduced cell. As another example, northern blot hybridization might be performed to compare the relative levels of a particular mRNA (and hence the expression level of the gene in question) among different tissues of an organism. Because an excess of DNA probe is used in these assays, the amount of hybridization is related to the amount of mRNA present in the original sam- ple, allowing the relative amounts of mRNA to be determined.

How are RNA molecules separated?

By using Gel E.; RNA has a negative charge but RNA molecules are usually single stranded. Since RNA molecules can make secondary and titer structures Glyoxal is used to glyoxylate the RNA therefore they can no make the 2nd and 3rd degree structures. By doing this they can track the RNA molecules based off of molecular weight and no the form of the molecule.

How does Circular DNA behave in Gel E?

Circular DNA is relaxed or nicked is fake af and it takes longer than linear DNA to migrate. The Circular DNA takes longer because it is not super coiled. Super coiled DNA molecules migrate faster through the gel because SMALL FRAGMENTS ALWAYS MOVE FASTER

Restriction Endonucleases

Cleaves DNA at particular sites by recognizing specific sequences.

How to construct DNA lib

Construction and probing of a DNA library. To construct the library, genomic DNA and vector DNA, digested with the same restriction enzyme, are incubated together with ligase. The resulting pool or library of hybrid vectors (each vector carrying a different insert of genomic DNA, represented in a different color) is then introduced into E. coli, and the cells are plated onto a filter placed over agar medium. Once colonies have grown, the filter is removed from the plate and prepared for hybridization: cells are lysed, the DNA is denatured, and the filter is incubated with a labeled probe. The clone of interest is identified by autoradiography

What share does DNA have and why can it migrate through the Gel E.?

DNA is negatively charged and when subjected to charge through Gel E. the DNA migrates towards the positive pole. electrode (anode) is what the pole is called

Dideoxynucleotides

Dideoxynucleotides used in DNA sequencing. On the left is 2'-deoxy ATP. This can be incorporated into a growing DNA chain and allow another nucleotide to be incorporated directly after it. On the right is 2',3'-dideoxy ATP. This can be incorporated into a growing DNA chain, but once in place it blocks further nucleotides being added to the same chain.

Genomic Libraries

Different kinds of libraries are made using insert DNA from different sources. The simplest are derived from total genomic DNA cleaved with a restriction enzyme; these are called genomic libraries.This type of library is most useful when generating DNA for sequencing a genome. If, on the other hand, the objective is to clone a DNA fragment encoding a particular gene, then a genomic library can be used efficiently only when the organism in question has relatively little non-coding DNA. For an organism with a more complex genome, this type of library is not suitable for this task because many of the DNA inserts will not contain coding DNA sequences.

Ethiduim

Dye that strains and fluoresces DNA molecules so it can be seen in the Gel E.

What does cleavage if an EcoRI site do?

EcoRI cuts the two strands within its recognition site to give 5' overhanging ends. These are called "sticky" ends — they readily adhere to other molecules cut with the same enzyme because they provide complementary single-strand ends that come together through base pairing.

PCR steps

Fig.7.12 Polymerase chain reaction (PCR). In the first step of the PCR, the DNA template is denatured by heating and annealed with synthetic oligonucleotide primers (dark orange and dark green) corresponding to the boundaries of the DNA sequence to be amplified. DNA polymerase is then used to copy the single-stranded template by extension from the primers (light orange and light green). In the next step, DNA is once again denatured, an- nealed with primers, and used as a template for a fresh round of DNA synthesis. Note that in this second cycle, the primers can prime synthesis from the newly synthesized DNAs as well as from the original template DNA. When DNA polymerase extends the green labeled primer that had annealed to newly synthesized (orange-labeled) template from the previous round of DNA synthesis (or orange-labeled primer from green- labeled template), the polymerase proceeds all the way to the end of the template and then falls off (in the figure [bottom], the polymerases have not yet reached the end of the templates). Thus, in this second cycle, DNA will have been synthesized that precisely spans the DNA sequence to be amplified. Thereafter, further rounds of denaturation, priming, and DNA synthesis (not shown) will generate DNAs that correspond to the sequence interval set by the two primers. This DNA will increase in abundance geometrically with each subsequent cycle of the chain reaction.

Site-directed mutagenesis

For example, a custom-designed oligonucleotide harboring a mismatch to a segment of cloned DNA can be used to create a directed mutation in that cloned DNA. This method, called site-directed mutagenesis, is performed as follows: the oligonucleotide is hybridized to the cloned DNA fragment and used to prime DNA synthesis with the cloned DNA as template. In this way, a double-strand molecule with one mismatch is made. The two strands are separated, and the strand with the desired mis- match is amplified further.

Can probes help with identification of separate DNA and RNA molecules?

Hybridization Probes Can Identify Electrophoretically Separated DNAs and RNAs

Libraries

In the remainder of this section, we describe how DNA molecules are cut, recombined, and propagated. We then discuss how large collections of such hybrid molecules, called libraries, can be created. In a library, a common vec- tor carries many alternative inserts. We describe how libraries are made and how specific DNA segments can be identified and isolated from them.

How does a southern blot work?

In this procedure , the cut DNA is separated by gel electrophoresis , and the gel is soaked in alkali to denature the double-stranded DNA fragments. These fragments are then transferred from the gel to a positively charged membrane to which they adhere, creating an imprint, or "blot," of the gel. During the transfer process, the DNA fragments are bound to the membrane in positions that mirror their corresponding positions in the gel after electrophoresis. After DNAs of interest are bound to the membrane, the charged membrane is incubated with a mixture of nonspecific DNA fragments to sat- urate all of the remaining binding sites on the membrane. Because the DNA in this mixture is randomly distributed on the membrane and, if chosen properly, will not contain the sequence of interest (e.g., from a different organism than the probe DNA), it will not interfere with subsequent detec- tion of a specific gene.

8. Describe the basis for separation of proteins for ion exchange , gel-filatrion and affinity column chromatography.

Ion-exchange chromatography separates proteins based on charge. Gel-filtration chromatography separates pro- teins based on size. Affinity chromatography separates proteins based on interaction with a specific molecule, protein, or nucleic acid that is coupled to the beads.

oligonucleotides

Many believe that the modern era of molecular biology was launched by the development of methods for the chemical synthesis of short, custom- designed segments of single-stranded DNA (ssDNA), known as oligonucleo- tides.

Plasmids and their function

Many common vectors are small (~3 kb) circular DNA molecules called plasmids. These molecules were originally derived from extrachromosomal circular DNA molecules that are found naturally in many bacteria and single-cell eukaryotes. In many cases (although not in yeast), these DNAs carry genes encoding resistance to antibiotics. Thus, naturally occurring plasmids already have two of the characteristics desirable for a vector: they can propagate independently in the host, and they carry a selectable marker. A further benefit is that these plasmids are sometimes present in multiple copies per cell. This increases the amount of DNA that can be isolated from a population of cells. Naturally occurring plasmids typically are restricted in the amount of DNA that can be carried (typically limited to 1-10 kb).

How does methylation effect Gel E?

Methylation of a base or enzymes that are sensitive to methylation can inhibit enzyme activity at the site.

5. Plasmid cloning vectors are specially designed to possess several features that are useful for cloning and expression. In a sentence or two, describe the role of each of the following features: origin of replication, restriction enzyme recognition sites, selectable marker, and promoter.

Origin of replication: Every plasmid requires an origin of replica- tion to serve as the start site of replication and to ensure trans- mission of the plasmid to the daughter cells after a cell division. Without replication, only one of the two daughter cells would carry the plasmid. Restriction enzyme recognition sites: Restriction enzyme sites can be used for insertion of a linear piece of DNA into the vec- tor by digestion and subsequent ligation. Selectable marker: A selectable marker (typically encoding a gene that confers resistance to an antibiotic) is used to identify which cells contain the vector carrying that marker. After transformation, cells are selected for the ability to grow in the presence of the antibiotic—only cells carrying the vector of interest survive under these conditions. Promoter: A promoter may be required for expression of the gene of interest carried on an expression vector. The promoter may be inducible and must be specific to—that is, active in—the host organism.

What are the two kind of Gel Matrices used?

Polyacrylamide and Agarose. Polyarcrylamide has high resolving and can separate DNA molecules only over a narrow range size. (<1000 bp) Agarose has less resolving power but can separate DNA molecules of up to serval hundred and thousands even kilo bases of bp.

Polymorphisms

Polymorphisms are alternative DNA se- quences (alleles) found in a population of organisms at a common, homologous region of the chromosome, such as a gene. A polymorphism can be as simple as alternative, single-base-pair differences at the same site in the chromosome among different members of the population or differences in the length of a simple nucleotide repeat sequence such as CA (see Chapter 9). What we want to do is amplify DNA surrounding and including the site of the polymorphism so that we can subject it to nucleotide sequencing (discussed later) and determine if there is a match to the sequence found in the crime scene sample. The nucleotide sequence of the amplified DNA helps to determine (along with checks for additional polymor- phisms) whether the two DNA samples match.

Probe

Probe is a defined sequence that is labeled and followed through mixtures to determine molecules that have a complimentary sequence , it is labeled so when it finds a target sequence it is easy located.

Why are restriction Enzymes used?

Restriction enzymes recognize typically ( 4-8 bp). They cut a defined position in the molecule within those sequences. Ex: If the enzymes recognizes " 5'GAATTC3' it cleaves the sequence at the places it recognizes the sequence at. EX: Linear DNA molecule with "GAATTC" in 6 places it enzyme is going to cleave in those spots creating 7 different segments that all with travel through the Gel E. differently based on size. Thus having cleaved the molecule into separate fragments that correspond to a particular region of the molecule. If we used a different enzymes we could cleave a different portion of the molecule for ex. AAGCTT (HindIII) which would Isolate different regions of the DNA molecule.

9.Explain the purpose of adding SDS to protein samples for polyacrylamide gel electrophoresis

SDS denatures the proteins by coating the proteins to create a uniform negative charge. This step allows the pro- teins to become separated by molecular weight in gel electrophoresis.

Linear DNA Molecules

Separate through the Gel according to size when subjected to a gel matrix

Genetic competence

Some bacteria, but not E. coli, can do this naturally and are said to have genetic competence. Competence is the ability of a cell to take up extracellular ("naked")

Expression vectors

Some vectors not only allow the isolation and purification of a particular DNA but also drive the expression of genes within the insert DNA. These plasmids are called expression vectors and have transcriptional promoters, derived from the host cell, immediately adjacent to the site of insertion. If the coding region of a gene (without its promoter) is placed at the site of inser- tion in the proper orientation, then the inserted gene will be transcribed into mRNA and translated into protein by the host cell. Expression vectors are frequently used to express heterologous or mutant genes to assess their function. They can also be used to produce large amounts of a protein for purification. In addition, the promoter in the expression vector can be chosen such that expression of the insert is regulated by the addition of a simple compound to the growth media (e.g., a sugar or an amino acid) This ability to control when the gene will be expressed is particularly useful if the gene product is toxic.

Contigs

Sophisticated computer programs have been developed that assemble the short sequences from random shotgun DNAs into larger contiguous sequences called contigs. Sequences or "reads" that contain identical sequences are assumed to overlap and are joined to form larger contigs (Fig. 7-18). The sizes of these contigs depend on the amount of sequence obtained — the more sequence, the larger the contigs and the fewer gaps in the sequence. Individual contigs are typically composed of 50,000-200,000 bp. This is still far short of a typical human chromosome. However, such contigs are useful for analyzing compact genomes.

Gel Electrophorsis

Tech. used to separte DNA and RNA molecules

DNA cloning

The ability to construct recombinant DNA molecules and maintain them in cells is called DNA cloning.

PCR

The game-changing method for amplifying particular segments of DNA, distinct from cloning and propagation within a host cell, is the polymerase chain reaction (PCR). This procedure is performed entirely biochemically, that is, in vitro. PCR uses the enzyme DNA polymerase that directs the syn- thesis of DNA from deoxynucleotide substrates on a single-stranded DNA template. As you will in Chapter 9, DNA polymerase adds nucleotides to the 3' end of a custom-designed oligonucleotide when it is annealed to a longer template DNA. Thus, if a synthetic oligonucleotide or primer is annealed to a single-strand template that contains a region complementary to the oli- gonucleotide, DNA polymerase can use the oligonucleotide as a primer and elongate its 3' end to generate an extended region of double-stranded DNA.

How does a Gel matrix function?

The gel matrix acts as sieve that the DNA molecules run through, the larger molecules have trouble moving through the pores compared to the small molecules thus larger molecules migrate more slowly through the gel. Large - slow Small - faster SMALL FRAGMENTS ALWAYS MOVE FASTER

phosphoamidines

The most common methods of chemical synthesis are performed on solid supports using machines that automate the process. The precursors used for nucleotide addition are chemically protected molecules called phosphoamidines (Fig. 7-11). In contrast to the direction of chain growth used by DNA polymerases (see Chapter 9), growth of the DNA chain is by addition to the 5' end of the molecule.

Chain-terminating nucleotides

The most commonly used procedure, which uses chain-terminating nucleotides and in vitro DNA synthesis, is the foundation for the original automation of DNA sequencing. In the chain-termination method, DNA is copied by DNA polymerase from a DNA template starting from a fixed point specified by hybridization of an oligonucleotide primer. DNA polymerase uses 2'-deoxynucleoside triphosphates as substrates for DNA synthesis, and DNA synthesis occurs by extending the 3' end. (The chain-termination method relies on the principles of enzymatic synthesis of DNA, which is discussed in Chapter 9.) The chain-termination method uses special, modi- fied substrates called 2',3'-dideoxynucleotides (ddNTPs), which lack the 3'-hydroxyl group on their sugar moiety as well as the 2'-hydroxyl (Fig. 7-13). DNA polymerase will incorporate a 2',3'-dideoxynucleotide at the 3' end of a growing polynucleotide chain, but once incorporated, the lack of a 3'-hydroxyl group prevents the addition of further nucleotides, causing elongation to terminate (Fig. 7-14).

BAC- bacterial artificial chromosome

The preceding results usually produce contigs that are <500 kb in length. These can be obtained using a special cloning vector called a BAC (bacterial artificial chromosome) that can accommodate very large inserts, up to hundreds of kilobases of DNA. A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. ... BACs are often used to sequence the genome of organisms in genome projects, for example the Human Genome Project. - Primers are used to obtain 600-bp sequencing reads from both ends of the BAC insert. These sequences are then aligned to different contigs, which can then be assigned to the same scaffold by virtue of sharing sequences from a common BAC insert. The use of BACs often permits the assignment of multiple contigs into a single scaffold of several megabases

Shotgun recombinant and shotgun sequencing

The process of producing "shotgun" recombinant libraries and huge excesses of random DNA-sequencing reads seems very wasteful. However, a cluster of 100 384-column automated sequencing machines can generate 10-fold coverage of a human chromosome in just a few weeks. This approach is considerably faster than the methods involving the isolation of known regions within the chromosome and sequentially sequencing a known set of staggered DNA fragments. Thus, the key technological insight that facili- tated the sequencing of the human genome was the reliance on automated shotgun sequencing and the subsequent use of computers to assemble the different pieces. The combination of automated sequencing machines and human genome (15 X 10 6 plasmids)

Reverse Transcription

The process that allows this is called reverse transcription and is performed by a special DNA polymerase (reverse transcriptase) that can make DNA from an RNA template (see Chap- ter 12). When treated with reverse transcriptase, mRNA sequences are con- verted into double-stranded DNA copies called cDNAs (for "copy DNAs"). From this point on, construction of the library follows the same strategy as does construction of a genomic library — the cDNA products and vector are treated with the same restriction enzyme, and the resulting fragments are then ligated into the vector.

Why is DNA cloning important?

This process typically involves a vector that provides the information necessary to propagate the cloned DNA in the replicat- ing host cell. Key to creating recombinant DNA molecules are the restriction enzymes that cut DNA at specific sequences and other enzymes that join the cut DNAs to one another. By creating recombinant DNA molecules that can be propagated in a host organism, a particular DNA fragment can be both purified from other DNAs and amplified to produce large quantities.

cDNA library

To enrich for coding sequences in the library, a cDNA library is created. This is made as shown in Figure 7-10. Instead of starting with genomic DNA, mRNAs are converted into DNA sequences. The process that allows this is called reverse transcription and is performed by a special DNA polymerase (reverse transcriptase) that can make DNA from an RNA template

DNA ligase

Treatment of the circle with the enzyme DNA ligase and ATP would seal the nicks to re-form a covalently closed circle. Seals nicks. DNA ligase is then used to link the compatible ends of the two DNAs. Adding an excess of the insert DNA rel- ative to the plasmid DNA ensures that the majority of vectors will reseal with insert DNA incorporated (Fig. 7-8).

What is hybridization?

Two molecules od DNA that go through the annealing process creating a base pairing of complementary strands.

How is this enzyme and reaction exploited to amplify specific DNA sequences?

Two synthetic, single-strand oligonucleotides are synthesized. One is complementary in sequence to the 5' end of one strand of the DNA to be amplified, and the other is complementary to the 5' end of the opposite strand (Fig. 7-12). The DNA to be amplified is then denatured, and the oligonucleotides are annealed to their target sequences. At this point, DNA polymerase and deoxynucleotide substrates are added to the reaction, and the enzyme extends the two primers. This reaction generates double-stranded DNA over the region of interest on both strands of DNA. Thus, two double-stranded copies of the starting fragment of DNA are pro- duced in this, the first, cycle of the PCR.

Autoradiogram

When a radioactively labeled probe DNA is used and X-ray film is exposed to the filter and then developed, an autoradiogram is produced in which the pattern of exposure on the film corresponds to the position of the hybrids on the blot

4. Compare and contrast the southern and northern blot

When performing a Southern blot, you detect a spe- cific DNA sequence with a DNA probe. When performing a northern blot, you detect a specific mRNA sequence with a DNA probe. When performing a Southern blot, you digest the genomic DNA with a restriction enzyme, separate the DNA frag- ments by gel electrophoresis, transfer the DNA to a positively charged membrane, and detect a fragment of DNA that contains your DNA of interest with the probe. You perform a similar set of steps for a northern blot except that you do not digest the mRNA population. In a northern blot, you can detect the amount of a cer- tain type of mRNA and can compare that to another sample pro- duced under different experimental conditions.

cDNAs

When treated with reverse transcriptase, mRNA sequences are converted into double-stranded DNA copies called cDNAs (for "copy DNAs").

What is the form of the Hpa1, EcoR1, Hind111, Pst1 ends?

generates blunt ends ( HA) and other like EcoR1,Hind111 and Pst1 generate staggered ends. Ecor1 cleaves covalent phosphdiester bonds between G and A at the starred positions on each strand (side note)

What is pulsed-felid gel electrophoresis?

pulsed - felid gel electrophoresis is how the size of large genomic DNA is determined. But.. how does it work? Well, the orientation of the long ass DNA molecule has to change its direction overtime when it experiences a pulse from the pulsed-felid gel electrophoresis machine (Duh). Hence the longer the strand takes to move the longer the DNA molecule is and thats how the size is determined.. yippy

Scaffold proteins

scaffold proteins are crucial regulators of many key signaling pathways. Although scaffolds are not strictly defined in function, they are known to interact and/or bind with multiple members of a signaling pathway, tethering them into complexes.

Shotgun sequencing Bacterial Genome/ 10X sequence coverage

small, compact genome that is composed of just 1.8 million base pairs (Mb) of DNA (less than l/1000th the size of the human genome). The H. influenzae genome was sheared into many random fragments with an average size of 1 kb. These pieces of genomic DNA were cloned into a plas- mid DNA vector to create a library. DNA was prepared from individual recombinant DNA colonies and separately sequenced on Sequenators using the dideoxy method discussed above. This method is called "shotgun" sequencing. Random recombinant DNA colonies are picked, processed, and sequenced. To ensure that every single nucleotide in the genome was captured in the final genome assembly, 30,000-40,000 separate recombi- nant clones were sequenced. A total of ~20 Mb of raw genome sequence was produced (600 bp of sequence is produced in an average reaction, and 600 bp x 33,000 different colonies = 20 Mb of total DNA sequence). This is called 10 x sequence coverage. In principle, every nucleotide in the genome should have been sequenced 10 times.

Transformation

transformation is the process by which a host organism can take up DNA from its environment.