Genetics Chapter 19: Molecular Genetic Analysis & Biotechnology

Replication in PCR has two essential requirements

(1) a DNA template from which a new DNA strand can be copied and (2) a pair of primers with a 3′-OH group to which new nucleotides can be added.

An effective cloning vector has three important characteristics, what are they?

(1) an origin of replication, which ensures that the vector is replicated within the cell; (2) selectable markers, which enable any cells containing the vector to be selected or identified; and (3) one or more unique restriction sites into which a DNA fragment can be inserted.

How do Northern and Western blotting differ from Southern blotting?

-Western blot is transferred from a protein gel. -Northern is RNA transferred and probed for the presence of specific sequences. -Southwestern blot is used to find where proteins (DNA binding proteins) bind to specific sequences of DNA.

1 map unit corresponds to how many base pairs?

1 million base pairs

How is Southern Blotting carried out?

1. DNA is first cleaved into fragments with restriction endonucleases. The fragments are separated by size via gel electrophoresis. 2. These fragments are then denatured and transferred by blotting onto the surface of a membrane filter. 3. The membrane filter now has single-stranded DNA fragments bound to its surface, separated by size as in the gel. 4. The filter is then incubated with a solution containing a denatured, labeled probe DNA. 5. The probe DNA hybridizes to its complementary DNA on the filter. 6. After washing away excess unbound probes, the labeled probe hybridized to the DNA on the filter can be detected using the appropriate methods to visualize the label. For radioactively labeled probes, the bound probe is detected by exposure to X-ray film. Other probe labeling methods detect bound probe using enzymatic reactions that generate luminescence or color.

The dideoxy method of DNA sequencing is based on the termination of DNA synthesis

1. Each of 4 reactions contains: (1) single-stranded target DNA to be sequenced, (2) a primer, (3) all 4 deoxyribonuceoside triphosphates, DNA polymerase, (4) and one type of dideocyribonucleoside triphosphate. 2. Nucleotides are added to the 3' end of the primer, with the target DNA being used as a template 3. When a dideoxynucleotide is incorporated into the growing chain, synthesis terminates because the dideoxynucleotide lacks a 3' OH 4. Synthesis terminates at different positions on different strands, which generates a set of DNA fragments of various lengths, each ending in a dideoxynucleotide with the same base 5. The fragments produced in each reaction are separated by gel electrophoresis 6. The sequence can be read directly from the bands that appear on the autoradiograph of the gel, starting from the bottom 7. The sequence obtained is the complement of the original template strand

lacZ gene can be used to screen bacteria containing recombinant plasmids

1. Foreign DNA is inserted into the partial lacZ gene 2. Bacteria that are lacZ- are transformed by the plasmid 3. Bacteria with an original (nonrecombinant) plasmid produce beta-galactosidase, which cleaves X-gal and makes the colonies blue 4. Bacteria with a recombinant plasmid do not synthesize beta-galactosidase. Their colonies remain white 5. Bacteria without a plasmid will not grow

positional cloning, case study

1. Linkage analysis carried out on families with CF 2. an association was found between the inheritance of molecular markers from chromosomes 3. Linkage studies with additional markers indicated CF locus close to specific markers 4. Clones from the region were isolated by chromosome walking and jumping 5. Analysis of DNA sequences within clones revealed 4 candidate genes 6. Additional studies eliminated 3 7. DNA sequencing revealed the presence of a 3-bp deletion in the gene of CF patient -expressed in pancreatic, respiratory, and sweat-gland tissues (all affected by the disease)

Creating a genomic library

1. Multiple copies of genomic DNA are digested by a restriction enzyme for a limited time so that only some of the restriction sites in each molecule are cut 2. Different DNA molecules are cut in different places, providing a set of overlapping fragments 3. Each fragment is then joined to a cloning vector 4. and transferred to a bacterial cell 5. ..producing a set of clones containing overlapping genomic fragments, some of which may include segments of the gene of interest Some clones contain the entire gene of interest, others include part of the gene, and most contain none of the gene of interest.

What are the steps to screening?

1. Plate the clones of the library 2. A disc of nitrocellulose or other membrane is laid on top of the bacterial colonies 3. A few cells from each colony ahere to the nitrocellulose filter 4. The cells are disrupted, and their DNA is denatured and fixed to the filter 5. A labeled probe hybridizes with any complementary DNA 6. Excess probe is washed off and the membrane is overlaid with X-ray film 7. ...which detects the presence of the probe 8. Comparison of the membrane with the master plate reveals which bacterial colonies have the DNA of interest

Steps to positional cloning

1. Use mapping studies to establish linkage between molecular markers and a phenotype of interest -linkage provides info about which chromosome carries the locus that codes for the phenotype and its general location on the chromosome 2. Use additional molecular markers clustered in the chromosomal region where the locus resides -more precisely locate the locus 3. Chromosome walking -start with a cloned gene marker close the the new gene of interest so the "walk" is shorter -neighboring genes are used to locate a gene of interest

What are the 2 additional advantages of a cDNA library

1. it is enriched with fragments from actively transcribed genes 2. introns do not interrupt the cloned sequences; introns would pose a problem when the goal is to produce a eukaryotic protein in bacteria, because most bacteria have no means of removing the introns.

Two key innovations facilitated the use of PCR in the laboratory

1. the discovery of a DNA polymerase that is stable at the high temperatures used in step 1 of PCR (DNA polymerase was isolated from the bacterium Thermus aquaticus, which lives in the boiling springs of Yellowstone National Park) -Taq polymerase 2. the development of automated thermal cyclers—machines that bring about the rapid temperature changes necessary for the different steps of PCR

limitations of PCR

1. the use of PCR requires prior knowledge of at least part of the sequence of the target DNA to allow the construction of the primers 2. the capacity of PCR to amplify extremely small amounts of DNA makes contamination a significant problem 3. accuracy (Taq polymerase does not have the capacity to proofread and, under standard PCR conditions, it incorporates an incorrect nucleotide about once every 20,000 bp. New heat-stable DNA polymerases with proofreading capacity have been isolated, giving more accurate PCR results.) 4. the size of the fragments that can be amplified by standard Taq polymerase is usually less than 2000 bp

challenges of working at a molecular level

1. very small 2. no physical features mark the beginning or end of a gene 3. separating the gene 4. inputting the new gene 5. making sure it is properly transcribed and translated 6. very poor ratios for successful take up and expression

A microbiologist discovers a new type II restriction endonuclease. When DNA is digested by this enzyme, fragments that average 1,048,500 bp in length are produced. What is the most likely number of base pairs in the recognition sequence of this enzyme?

4n = 1,048,500, so n = 10. A 10-bp recognition sequence is most likely.

Genetically engineered corn now constitutes ___ of all corn grown in the U.S.

88%

DNA library

A collection of clones containing all the DNA fragments from one source

reverse-transcription PCR

Amplifies sequences corresponding to RNA. Reverse transcriptase is used to convert RNA into complementary DNA, which can then be amplified by the usual polymerase chain reaction.

cDNA library

An alternative to creating a genomic library is to create a library consisting only of those DNA sequences that are transcribed into mRNA (called a cDNA library because all the DNA in this library is complementary to mRNA)

Why is the use of a heat-stable DNA polymerase important to the success of PCR?

Because the solution must be heated to break down the hydrogen bonds in the DNA, and you don't want the polymerase to break at this point

DNA fragments that are 500 bp, 1000 bp, and 2000 bp in length are separated by gel electrophoresis. Which fragment will migrate farthest in the gel? A. 2000-bp fragment B. 1000-bp fragment C. 500-bp fragment D. All will migrate equal distances.

C. 500-bp fragment, because it is the shortest and has the least hinderance

A DNA library can be screened for a specific gene with the use of complementary probes that hybridize to the gene. Alternatively, the library can be cloned into an expression vector, and the gene can be located by examining the clones for the protein product of the gene.

CONCEPT TO KNOW

DNA fragments can be inserted into cloning vectors, stable pieces of DNA that will replicate within a cell. A cloning vector must have an origin of replication, one or more unique restriction sites, and selectable markers. An expression vector contains sequences that allow a cloned gene to be transcribed and translated. Special cloning vectors have been developed for introducing genes into eukaryotic cells.

CONCEPT TO KNOW

DNA fragments can be separated, and their sizes can be determined with the use of gel electrophoresis. The fragments can be viewed by using a dye that is specific for nucleic acids or by labeling the fragments with a chemical tag.

CONCEPT TO KNOW

Labeled probes, which are sequences of RNA or DNA that are complementary to the sequence of interest, can be used to locate individual genes or DNA sequences. Southern blotting can be used to transfer DNA fragments from a gel to a membrane such as nitrocellulose.

CONCEPT TO KNOW

Molecular genetic analyses require special methods because individual genes make up a tiny fraction of the cellular DNA and they cannot be seen.

CONCEPT TO KNOW

Molecular genetics and recombinant DNA technology are used to locate, analyze, alter, study, and recombine DNA sequences. These techniques are used to probe the structure and function of genes, address questions in many areas of biology, create commercial products, and diagnose and treat diseases.

CONCEPT TO KNOW

One method of finding a gene is to create and screen a DNA library. A genomic library is created by cutting genomic DNA into overlapping fragments and cloning each fragment in a separate bacterial cell. A cDNA library is created from mRNA that is converted into cDNA and cloned in bacteria.

CONCEPT TO KNOW

Positional cloning allows researchers to isolate a gene without having knowledge of its biochemical basis. Linkage studies are used to map the locus producing a phenotype of interest to a particular chromosome region. Chromosome walking and jumping can be used to progress from molecular markers to clones containing sequences that cover the chromosome region. Candidate genes within the region are then evaluated to determine if they encode the phenotype of interest

CONCEPT TO KNOW

The polymerase chain reaction is an enzymatic, in vitro method for rapidly amplifying DNA. In this process, DNA is heated to separate the two strands, short primers attach to the target DNA, and DNA polymerase synthesizes new DNA strands from the primers. Each cycle of PCR doubles the amount of DNA. PCR has a number of important applications in molecular biology.

CONCEPT TO KNOW

Type II restriction enzymes cut DNA at specific base sequences that are palindromic. Some restriction enzymes make staggered cuts, producing DNA fragments with cohesive ends; others cut both strands straight across, producing blunt-ended fragments. There are fewer long recognition sequences in DNA than short recognition sequences.

CONCEPT TO KNOW

expression vector

Cloning vector containing DNA sequences such as a promoter, a ribosome-binding site, and transcription initiation and termination sites that allow DNA fragments inserted into the vector to be transcribed and translated

cosmid

Cloning vector that combines the properties of plasmids and phage vectors and is used to clone large pieces of DNA in bacteria. A cosmid is a small plasmid that carries a λ cos site, allowing the plasmid to be packaged into a viral coat

Give three important characteristics of cloning vectors

Cloning vectors should have: (1) An origin of DNA replication so they can be maintained in a cell (2) A gene, such as antibiotic resistance, to select for cells that carry the vector (3) A unique restriction site or series of sites to where a foreign DNA molecule may be inserted

genomic library

Collection of bacterial or phage colonies containing DNA fragments that consist of the entire genome of an organism

genetic engineering

Common term for recombinant DNA technology

_______ was the first genetic disease for which the causative gene was isolated entirely by positional cloning

Cystic fibrosis

After DNA fragments have been separated by gel electrophoresis, how can they be visualized?

DNA molecules can be visualized by staining with a fluorescent dye, such as ethidium bromide, that intercalates between the stacked bases of the DNA double helix, and the dye-DNA complex fluoresces when irradiated with an ultraviolet light source. Alternatively, they can be visualized by attaching radioactive or chemical labels to the DNA before it is placed in the gel.

Taq polymerase

DNA polymerase commonly used in PCR reactions. Isolated from the bacterium Thermus aquaticus, the enzyme is stable at high temperatures, and so it is not denatured during the strand-separation step of the cycle.

Explain how gel electrophoresis is used to separate DNA fragments of different lengths.

Gel electrophoresis uses an electric field to drive DNA molecules through a gel that acts as a molecular sieve. The gel is an aqueous matrix of agarose or polyacrylamide. 1. DNA molecules are loaded into a slot or well at one end of the gel. 2. When an electric field is applied, the negatively charged DNA molecules migrate toward the positive electrode. 3. Shorter DNA molecules are less hindered by the agarose or polyacrylamide matrix and migrate faster than longer DNA molecules, which must wind their way around obstacles and through the pores in the gel matrix. 4. A dye specific for nucleic acids is added 5. DNA fragments appear as bands on the gel

gene cloning

Insertion of DNA fragments into bacteria in such a way that the fragments will be stable and copied by the bacteria -original copies (clones) of the original piece of DNA are produced

Often, real-time PCR is combined with reverse-transcription PCR to measure the amount of mRNA in a sample, allowing biologists to determine the level of gene expression in different cells and under different conditions

JUST TO KNOW

probe

Known sequence of DNA or RNA that is complementary to a sequence of interest and will pair with it; used to find specific DNA sequences.

Ti plasmid

Large plasmid isolated from the bacterium Agrobacterium tumefaciens and used to transfer genes to plant cells

Genetic engineering is being used to treat what form of genetic blindness?

Leber congenital amaurosis there is a mutation in one of a number of genes responsible for the development or function of light receptors in the retina. LCA is inherited as an autosomal recessive disorder; in people with two defective copies of a gene, the light-sensing cells die. Children with LCA begin losing sight at birth, and most are completely blind by age 40.

chromosome walking

Method of locating a gene by using partly overlapping genomic clones to move in steps from a previously cloned, linked gene to the gene of interest -basis of chromosome walking is the fact that a genomic library consists of a set of overlapping DNA fragments

positional cloning

Method that allows for the isolation and identification of a gene by examining the cosegregation of a phenotype with previously mapped genetic markers

in situ hybridization

Method used to determine the chromosomal location of a gene or other specific DNA fragment OR the tissue distribution of an mRNA by using a labeled probe that is complementary to the sequence of interest

real-time PCR

Modification of the polymerase chain reaction that quantitatively determines the amount of starting nucleic acid; the amount of DNA amplified is measured as the reaction proceeds

the Molecular Genetics Revolution

Previously, information about the structure and organization of genes was gained by examining their phenotypic effects, but molecular genetic analysis allows the nucleotide sequences themselves to be read. -replication -transcription -translation -RNA processing -gene regulation

Southern blotting

Process by which DNA is transferred from a gel to a solid support such as a nitrocellulose or nylon filter.

Northern blotting

Process by which RNA is transferred from a gel to a solid support such as a nitrocellulose or nylon filter.

DNA sequencing

Process of determining the sequence of bases along a DNA molecule

restriction enzyme

Recognizes particular base sequences in DNA and makes double-stranded cuts nearby; also called restriction endonuclease.

cohesive ends ("sticky ends")

Short, single-stranded overhanging end on a DNA molecule produced when the DNA is cut by certain restriction enzymes. Cohesive ends are complementary and can spontaneously pair to rejoin DNA fragments that have been cut with the same restriction enzyme.

linker

Small, synthetic DNA fragment that contains one or more restriction sites. Can be attached to the ends of any piece of DNA and used to insert a gene into a plasmid vector when restriction sites are not available.

What is the purpose of Southern blotting?

Southern blotting is used to detect and visualize specific DNA fragments that have a sequence complementary to a labeled DNA probe.

cloning vector

Stable, replicating DNA molecule to which a foreign DNA fragment can be attached and transferred to a host cell

restriction endonuclease

Technical term for a restriction enzyme, which recognizes particular base sequences in DNA and makes double-stranded cuts nearby.

gel electrophoresis

Technique for separating charged molecules (such as proteins or nucleic acids) on the basis of molecular size or charge or both.

Application: crops

The Bt toxin gene, which encodes an insecticide, was isolated from bacteria and transferred to tobacco plants.

How are candidate genes that are identified by positional cloning evaluated to determine whether they encode the phenotype of interest?

The expression pattern of the gene—where and when it is transcribed—can often provide clues about its function.

Suppose that a geneticist discovers a new restriction enzyme in the bacterium Aeromonas ranidae. This restriction enzyme is the first to be isolated from this bacterial species. Using the standard convention for abbreviating restriction enzymes, give this new restriction enzyme a name (for help, see footnote to Table 19.1).

The first three letters are taken from the genus and species name, and the Roman numeral indicates the order in which the enzyme was isolated. Therefore, the enzyme should be named AraI.

What feature is commonly seen in the sequences recognized by type II restriction enzymes?

The recognition sequences are palindromic, and 4−8 base pairs long.

biotechnology

Use of biological processes, particularly molecular genetics and recombinant DNA technology, to produce products of commercial value.

About 60% of the base pairs in a human DNA molecule are AT. If the human genome has 3.2 billion base pairs of DNA, about how many times will the following restriction sites be present? a. BamHI (restriction site is 5′-GGATCC-3′) b. EcoRI (restriction site is 5′-GAATTC-3′) c. HaeIII (restriction site is 5′-GGCC-3′)

We must first calculate the frequency of each base. Given that AT base pairs consist 60% of the DNA, we deduce that the frequency of A is 0.3 and frequency of T is 0.3. The GC base pairs must consist of 40% of the DNA; therefore, the frequency of G is 0.2 and the frequency of C is 0.2. BamH1 GGATCC is then (0.2)(0.2)(0.3)(0.3)(0.2)(0.2) = 0.000144 3,200,000,000(0.000144) = 460,800 times EcoRI GAATTC = (0.2)(0.3)(0.3)(0.3)(0.3)(0.2) = 0.000324 3,200,000,000(0.000324) = 1,036,800 times HaeIII GGCC = (0.2)(0.2)(0.2)(0.2) = 0.0016 3,200,000,000(0.0016) = 5,120,000 times

yeast artificial chromosome (YAC)

a DNA molecule that has a yeast origin of replication, a pair of telomeres, and a centromere. These features ensure that YACs are stable, replicate, and segregate in the same way as yeast chromosomes. YACs are particularly useful because they can carry DNA fragments as large as 600 kb, and some special YACs can carry inserts of more than 1000 kb. YACs have been modified so that they can be used in eukaryotic organisms other than yeast.

Briefly explain how synthetic probes are created to screen a DNA library when the protein encoded by the gene is known.

a set of DNA probes can be synthesized chemically by using an automated machine known as an oligonucleotide synthesizer.

Recombinant DNA technology

a set of molecular techniques for locating, isolating, altering, and studying DNA segments

Which vectors (plasmid, phage λ, cosmid, bacterial artificial chromosome) can be used to clone a continuous fragment of DNA with the following lengths? a. 4 kb b. 20 kb c. 35 kb d. 100 kb

a. 4 kb - plasmid b. 20 kb - λ phage c. 35 kb - cosmid d. 100 kb - bacterial artificial chromosome

chromosome jumping

allows one to move from more distantly linked markers to clones that contain a sequence of interest

What are some uses for genetic engineering?

bacteria that generate chemicals, pest-resistant crops, and farm animals that secrete pharmaceutical products in their milk

shotgun cloning

clone first and search later -a researcher first clones a large number of DNA fragments, knowing that one or more contains the DNA of interest, and then searches for the fragment of interest among the clones.

fragments with short, single-stranded overhanging ends

cohesive ends

PvuII

cuts the DNA straight across, producing blunt ends

HindIII

cuts the sugar-phosphate backbone of each strand in a staggered fashion, generating fragments with short, single-stranded overhanging ends

With in situ hybridization, DNA probes are used to...

determine the cellular or chromosomal location of a gene or its product.

Allan Maxam and Walter Gilbert Method

dideoxy-sequencing method based on chemical degradation of DNA

Sanger (dideoxy) Method

dideoxy-sequencing method based on the elongation of DNA by DNA polymerase -replication -In the process, replication is sometimes (but not always) terminated when a specific base is encountered, producing DNA strands of different lengths, each of which ends in the same base. -special substrate (dideoxyribonucleoside triphosphate) ---> that they lack a 3′-OH group

The polymerase chain reaction (PCR)

first developed in 1983 by Kary Mullis, allows DNA fragments to be amplified a billionfold within just a few hours. It can be used with extremely small amounts of original DNA, even a single molecule. The polymerase chain reaction has revolutionized molecular biology and is now one of the most widely used molecular techniques.

Gene therapy

genetic engineering used to treat disease

Recomninant

goal is to combine DNA from 2 distinct sources

What is the disadvantage of a cDNA?

it contains only sequences that are present in mature mRNA. Sometimes, researchers are interested in sequences that are not transcribed, such as those in promoters and enhancers, which are important for transcription but are not themselves transcribed. These sequences are not present in a cDNA library.

engineered nucleases

more complex enzymes that are capable of making double-stranded cuts to the DNA at any predetermined DNA sequence -consist of part of a restriction enzyme that cleaves the DNA, coupled with another protein that recognizes and binds to a specific DNA sequence; the particular sequence to which the protein binds is determined by the protein's amino acid sequence. -can be custom designed to bind to and cut any particular DNA sequence

Where do restriction enzymes come from?

produced naturally by bacteria

palindromic sequences

read the same (5' to 3') on the two complementary DNA strands

can be used to quantitatively determine the amount of starting nucleic acid

real-time PCR

Type II restriction enzymes

recognize specific sequences and cut the DNA at defined sites within or near the recognition sequence virtually all molecular genetics is done with this type

The basis of PCR is

replication catalyzed by a DNA polymerase

These enzymes are produced naturally by bacteria and are used in defense against viruses

restriction enzyme A bacterium protects its own DNA from a restriction enzyme by modifying the recognition sequence, usually by adding methyl groups to its DNA.

DNA ligase

seals nicks between the sugar-phosphate groups of the fragments

What has decreased the use of RFLPs?

the availability of inexpensive DNA sequencing technology (PCR) has decreased the use of RFLPs in gene mapping and genetic diagnosis

Genetic engineering

the manipulation and transfer of genes from one organism to another

Western blotting

the transfer of protein from a gel to a membrane. Here, the probe is usually an antibody, used to determine the size of a particular protein and the pattern of the protein's expression.

relation between the length of the recognition sequence and the number of times that it is present in a genome

there will be fewer longer recognition sequences than shorter recognition sequences -restriction enzymes that recognize longer sequences will cut a given piece of DNA into fewer and longer fragments than will restriction enzymes that recognize shorter sequences.

restriction fragment length polymorphisms (RFLPs)

variations (polymorphisms) in the patterns of fragments produced when DNA molecules are cut with the same restriction enzyme (Figure 19.22). These differences are inherited and can be used in mapping, similar to the way in which allelic differences are used to map conventional genes.

Bacterial artificial chromosomes (BACs)

vectors originally constructed from the F plasmid (a special plasmid that controls mating and the transfer of genetic material in some bacteria; see Chapter 9) and can hold very large fragments of DNA that can be as long as 300,000 bp. Table 19.2 compares the properties of plasmids, phage λ vectors, cosmids, and BACs.

The screening procedure used to find clones that contain the gene of interest depends on what?

what is known about the gene