Chapter 12- Modern applications of microbial genetics

Question 1

Biotechnology

Answer 1

The science of using living systems to benefit humankind. It is associated with genetic engineering

Question 2

Genetic engineering

Answer 2

The direct alteration of an organism’s genetics to achieve desirable traits. It involves recombinant DNA technology

Question 3

Recombinant DNA technology

Answer 3

The process by which a DNA sequence is manipulated in vitro, creating recombinant DNA molecules that have new combinations of genetic material. The recombinant DNA is then introduced into a host organism

Question 4

Transgenic

Answer 4

An organism that has recombinant DNA that was introduced from a different species. One example is the bacterial strain that produces human insulin

Question 5

How is insulin produced?

Answer 5

The insulin gene from humans is inserted into a bacterial plasmid. The recombinant DNA plasmid was then inserted into bacteria, making it transgenic. The bacteria can then produce and secrete human insulin

Question 6

Molecular cloning

Answer 6

A set of methods used to construct recombinant DNA and incorporate it into a host organism

Question 7

Restriction endonucleases

Answer 7

Restriction enzymes- bacterial enzymes that are produced as a defense mechanism to cut and destroy foreign cytoplasmic DNA that typically occurs due to bacteriophage infection. In biotechnology, they are used to cut DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules

Question 8

Recognition site

Answer 8

A specific palindromic DNA sequence that is between 4 and 6 base pairs. A restriction enzyme recognizes the DNA palindrome and cuts each DNA backbone at identical positions in the palindrome

Question 9

Sticky/blunt ends

Answer 9

Some restriction enzymes cut to produce molecules that have complementary overhangs called sticky ends. Others cut without producing overhangs, making blunt ends

Question 10

Hybridization

Answer 10

Refers to the joining together of two complementary single strands of DNA. This can be done more easily in molecules with sticky ends, because the sticky ends can anneal

Question 11

Anneal

Answer 11

Form hydrogen bonds between complementary bases (at their sticky ends)

Question 12

Ligation

Answer 12

When DNA ligase rejoins the two sugar phosphate backbones of DNA through covalent bonding, making the molecule a continuous double strand

Question 13

Vectors

Answer 13

DNA molecules that carry DNA fragments from one organism to another. Plasmids are used as vectors and can be genetically engineered to have specific properties

Question 14

Polylinker site

Answer 14

A short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both the DNA and the plasmid. Multiple restriction enzyme recognition sites in the polylinker site makes the plasmid vector versatile, and it can be used for multiple different cloning experiments

Question 15

Reporter gene

Answer 15

A gene sequence that is artificially engineered into the plasmid. It encodes a protein that allows for visualization of DNA insertion. Researchers use it to determine which cells are host cells with recombinant plasmids and which contain the non-recombinant plasmid vector. The polylinker site is often found within a reporter gene

Question 16

Transformation

Answer 16

A process in which bacteria take up free DNA from their surroundings. In nature, free DNA comes from other lysed bacterial cells

Question 17

Molecular cloning using transformation

Answer 17

In the laboratory, free DNA is the form of recombinant plasmids is introduced to the cell’s surrounding. Some bacteria are naturally competent and are able to take up foreign DNA. Bacteria can also be made artificially competent by increasing the permeability of the cell membrane. This is done through chemical treatments or through exposing the bacteria to an electric field that creates microscopic pores in the cell membrane. After transformation protocol, bacterial cells are put into a medium containing antibiotics to inhibit the growth of host cells that didn’t get the antibiotic resistance plasmid

Question 18

Molecular cloning using conjugation

Answer 18

F plasmids are transferred between bacterial cells using conjugation. Recombinant DNA is transferred through conjugation when bacterial cells containing a recombinant F plasmid are mixed with bacterial cells that lack the plasmid. The F pilus is encoded by the plasmid and forms a bridge between the cells. The F plasmid containing cell replicates its plasmid and transfers a copy of the recombinant F plasmid to the recipient cell. Once it has received the recombinant F plasmid, the recipient cell can produce its own F pilus and facilitate the transfer of the recombinant F plasmid to an additional cell.

Question 19

Molecular cloning using transduction

Answer 19

DNA fragments of interest are engineered into phagemids. Bacterial cells can then be infected with these bacteriophages so that the recombinant phagemids can be introduced into the bacterial cells. Depending on the type of phage, the recombinant DNA can be integrated into the host genome or it can exist as a plasmid in the host’s cytoplasm

Question 20

Phagemids

Answer 20

Plasmids that have phage sequences that allow them to be packaged into bacteriophages

Question 21

Genomic library

Answer 21

A complete copy of an organism’s genome contained as recombinant DNA plasmids, engineered into unique clones of bacteria. Researchers can create large quantities of each fragment by growing the bacterial host for that fragment

Question 22

How are genomic libraries created?

Answer 22

By ligating individual restriction enzyme digested genomic fragments into plasmid vectors that have been cut with the same restriction enzyme. Each bacterial cell has a single recombinant plasmid and grows into a colony of cells. All of the cells in this colony are identical clones and carry the same recombinant plasmid. The resulting library is a collection of colonies, each of which contains a fragment of the original organism’s genome, that are each separate and distinct and can each be used for further study.

Question 23

Complementary DNA (cDNA)

Answer 23

All cells have the same DNA, but only certain genes are expressed in certain tissues, and only the genes that are expressing a trait are synthesizing relevant mRNA. mRNA can’t be cloned directly, so in the laboratory, mRNA is used as a template by the reverse transcriptase enzyme to make cDNA. A cell’s full complement of mRNA can be reverse transcribed into cDNA molecules, which can be used as a template for DNA polymerase to make double stranded DNA copies, and can be used to make a cDNA library.

Question 24

What is the benefit of a cDNA library?

Answer 24

The benefit of a cDNA library is that it contains DNA from only the expressed genes in the cell. This means that the introns, control sequences such as promoters, and DNA not destined to be translated into proteins are not represented in the library.

Question 25

Transfection

Answer 25

The introduction of recombinant DNA molecules into eukaryotic hosts, mainly plants and animals

Question 26

Why is transfection of eukaryotic hosts more challenging?

Answer 26

Eukaryotes aren’t competent to take up foreign DNA and they can’t maintain plasmids like bacteria are able to. Plant cells are also more difficult to transfect than animal cells because they have thick cell walls

Question 27

Electroporation

Answer 27

An electric pulse of a cell culture induces the formation of transient pores in the phospholipid bilayers of cells through which the gene can be introduced. At the same time, the electric pulse generates a brief positive charge on one side of the cell and a negative charge on the opposite side. The charge difference draws negatively charged DNA molecules into the cell. This is a method of transfection in eukaryotic cells

Question 28

Microinjection

Answer 28

Another method of transfection. Eukaryotic cells are larger than prokaryotic cells, so DNA fragments can sometimes be directly injected into the cytoplasm using a micropipette

Question 29

Gene guns

Answer 29

A method of transfecting plant cells. Plant cells are treated with enzymes to remove their cell walls. Then, a gene gun is used to shoot gold or tungsten particles coated with recombinant DNA molecules into the plant protoplasts. Recipient protoplast cells can then recover and be used to generate new transgenic plants

Question 30

Shuttle vectors

Answer 30

Plasmids that can move between bacterial and eukaryotic cells

Question 31

Tumor inducing (Ti) plasmids

Answer 31

Originate from bacteria and are used as shuttle vectors to incorporate genes into plants. These plasmids naturally occur and cause tumors when they are transferred from bacterial cells to plant cells. Researchers can manipulate them to remove their tumor causing genes and insert desirable DNA fragments. The plasmids then naturally transfer from the bacterium to the plant cell and then recombine into the plant cell’s genome

Question 32

Viral vectors

Answer 32

Used to transfect eukaryotic cells and are often used in gene therapy to introduce healthy genes. Viral genes can be depleted and replaced with the gene to be delivered. The virus then infects the host cell and delivers the foreign DNA into the genome of the targeted cell. Adenoviruses are often used for this purpose because they can be grown to high titer and can infect both nondividing and dividing host cells

Question 33

Protein signatures

Answer 33

The expression levels of specific arrays of proteins. Protein signatures can be compared to evaluate cellular responses to a multitude of environmental factors and stresses. They can also reveal the identity of organism or how a cell is responding during disease

Question 34

DNA probe

Answer 34

Used to identify the presence of a certain DNA sequence. It uses artificially single stranded constructed pieces of DNA (probes). It is used to screen for a gene of interest in a stretch of DNA. The probes are complementary to the gene of interest and different from other DNA sequences in the sample. The probe has to be labeled with a molecular tag or beacon, like a radioactive phosphorus atom or a fluorescent dye so that the probe and the DNA it binds to can be seen. The DNA sample also needs to be denatured so it will be single stranded and the probe can bind to it

Question 35

Autoradiography

Answer 35

When a DNA probe is used to screen for a gene of interest. The probe is labeled with a radioactive phosphorus atom

Question 36

Fluorescent in situ hybridization (FISH)

Answer 36

When a DNA probe is used to screen for a gene of interest. The probe is labeled with a fluorescent dye

Question 37

Agarose gel electrophoresis

Answer 37

Used to separate DNA or RNA of varying sizes that may be generated by restriction enzyme digestion or by other means, like PCR. The gel is oriented horizontally in a buffer solution

Question 38

How does agarose gel electrophoresis work?

Answer 38

The sugar-phosphate backbone of DNA is negatively charged, so DNA is attracted to a positive electrode. Samples are placed in sample well on the side of the gel that is closest to the negative electrode. It is then drawn through the agarose matrix toward the positive electrode. Smaller molecules are able to pass through more quickly, while larger molecules are impeded by the gel. Therefore, smaller molecules can travel a longer distance. Sizes of DNA fragments within a sample can be estimated by comparison to fragments of known size in a DNA ladder also run on the same gel.

Question 39

Pulsed-field gel electrophoresis

Answer 39

A variation of agarose gel electrophoresis used to separate very large DNA fragments like chromosomes or viral genomes. The orientation of the electric field is periodically alternated. Smaller fragments can reorient themselves and migrate slightly faster than larger fragments. Therefore, very large fragments that would otherwise travel together can be separated

Question 40

How are DNA and RNA fragments visualized during gel electrophoresis?

Answer 40

Ethidium bromide is a stain that inserts into nucleic acids at non-specific locations can be visualized when exposed to UV light

Question 41

Restriction enzyme recognition sites

Answer 41

Short sequences that are only a few nucleotides long. They are sequence-specific palindromes that can be found throughout the genome. Differences in DNA sequences in the genomes of individuals will lead to differences in distribution of restriction enzyme recognition sites.

Question 42

Restriction fragment length polymorphism (RFLP) analysis

Answer 42

Differences in individual distribution of restriction enzyme recognition sites can be visualized as distinct banding patterns on a gel after agarose gel electrophoresis. It can be used on human genomes to determine inheritance patterns of chromosomes with variant genes, and can therefore determine paternity. It can also be used by forensic scientists to analyze DNA obtained from crime scenes, suspects, or victims

Question 43

Southern blot technique

Answer 43

DNA fragments in a sample are separated by gel electrophoresis and then transferred to a membrane through capillary action. The DNA fragments that bind to the surface of the thin positively charged membrane are then exposed to a specific single stranded DNA probe that is labeled using a radioactive molecule or fluorescent dye so it can be visualized. The membrane prevents nucleic acids from diffusing out in the gel. Southern blots can be used to detect the presence of certain DNA sequences within a sample. Once the DNA sequence is visualized, researchers can cut out the portion of the membrane containing the fragment to isolate it

Question 44

Colony blot

Answer 44

Another variation of the Southern blot where colonies representing different clones in a genomic library are transferred to a membrane. The cells on the membrane are lysed and the membrane can then be probed to determine which colonies in the genomic library harbor the target gene

Question 45

Northern blot

Answer 45

A variation of the Southern blot where RNA is immobilized on the membrane and probed. This technique is used to detect the amount of mRNA made through gene expression within a tissue or organism sample

Question 46

Microarray analysis

Answer 46

Used to compare gene expression patterns between different cell types, like cancerous or virus infected cells with healthy cells. DNA or cDNA from a sample is placed on a glass slide along known DNA sequences, and cDNA or DNA can be spotted on a glass slide. Once they are deposited, genomic DNA or mRNA can be isolated from the two samples for comparison. If
mRNA is isolated, it is reverse-transcribed to cDNA using reverse transcriptase. Then the two samples of
genomic DNA or cDNA are labeled with different fluorescent dyes (typically red and green). The labeled genomic DNA samples are allowed to hybridize to complementary spots on the microarray

Question 47

How is hybridization of sample genomic DNA molecules monitored during the microarray analysis technique?

Answer 47

The intensity of fluorescence at particular spots on the microarray is measured. Differences in the amount of hybridization between the samples can be readily observed. If only one sample’s nucleic acids hybridize to a particular spot on the microarray, then that spot will appear either green or red. However, if both samples’ nucleic acids hybridize, then the spot will appear yellow due to the combination of the red and green dyes

Question 48

Drawbacks of microarray technology

Answer 48

It requires sophisticated and expensive detection equipment and analysis software. Therefore, the use of this technology is typically limited to research settings

Question 49

Polyacrylamide gel electrophoresis (PAGE)

Answer 49

Used for separating proteins. The gel matrix is composed of polyacrylamide rather agarose, so it’s finer. It’s performed using a vertical gel apparatus. Different charges are associated with amino acid side chains, so PAGE can be used to separate proteins based on their net charges.

Question 50

SDS gel electrophoresis

Answer 50

Another version of PAGE. The proteins are denatured and coated with a negatively charged detergent (SDS) that masks the negative charges and allows separation of the proteins based on size rather than charge

Question 51

2D gel electrophoresis

Answer 51

Proteins are separated based on two characteristics, like separation of charges at various pHs as well as their size

Question 52

Polymerase chain reaction (PCR)

Answer 52

Allows for rapid amplification in the number of copies of specific DNA sequences. It can be used to determine the presence of difficult to culture microorganisms in humans, analyzing samples to determine paternity, identifying the source of a DNA sample left at a crime scene, amplifying a target region of DNA for cloning into a plasmid vector, or for determine the sequence of nucleotides in a specific region of DNA

Question 53

DNA primers

Answer 53

Primers used for PCR, which are preferred due to their stability. They are functionally similar to a DNA probe and bind to specific complementary targets. They are necessary because a 3’ hydroxyl group is needed for DNA polymerase

Question 54

Steps of PCR (3)

Answer 54

1. Denaturation
2. Annealing
3. Extension

Question 55

PCR process

Answer 55

A double stranded template of DNA that contains the target sequence is denatured at a high temperature (90), which physically separates the DNA strands. The temperature is then lowered to 50 degrees, so the DNA primers complementary to the ends of the target sequence anneal (stick) to the template strands. The temperature is then raised to 72, which is the optimal temperature for the activity of the heat stable DNA polymerase. This allows nucleotides to be added to the primer using the single stranded target as a template. One primer binds to each strand. Each cycle doubles the number of double-stranded target DNA copies. Typically, PCR protocols include 25–40 cycles, allowing for the amplification of a single target sequence by tens of millions to over a trillion

Question 56

Which machines are used for PCR?

Answer 56

Thermal cyclers. There are also isothermal PCR amplification methods that circumvent the need for thermal cycling- they take advantage of accessory proteins that aid in the DNA replication process

Question 57

Reverse transcriptase PCR (RT-PCR)

Answer 57

Used for obtaining DNA copies of a specific mRNA molecule. It can detect whether a specific gene has been expressed by a sample. The reverse transcriptase enzyme converts mRNA molecules into cDNA. The cDNA is used as a template for traditional PCR amplification

Question 58

Real time PCR

Answer 58

Also called quantitative PCR (qPCR). The use of fluorescence allows you to monitor the increase in a double stranded template during a PCR reaction as it occurs. The kinetics data can be used to quantify the amount of the original target sequence. It allows researchers to determine the number of DNA copies present in a sample. It can also be used to determine viral load in HIV positive patients to evaluate the effectiveness of their therapy

Question 59

Chain termination method

Answer 59

Also called the Sanger DNA sequencing method. It involves DNA replication of a single stranded template with the use of a DNA primer to initiate synthesis of a complementary strand. It also involves DNA polymerase, 4 regular deoxynucleotide monomers, and a small amount of dideoxynucleotides. When dideoxynucleotides are randomly incorporated into the growing complementary strand, it terminates the process of DNA replication for that particular strand. This results in multiple short strands of
replicated DNA that are each terminated at a different point during replication. The reaction mixture undergoes gel electrophoresis, and multiple newly replicated DNA strands form a ladder of differing sizes

Question 60

Dideoxynucleotides

Answer 60

Monomers missing a hydroxyl group at the site at which another nucleotide usually attaches to form a chain. In chain termination DNA sequencing, each ddNTP is labeled with a different colored fluorescent dye or fluorochrome, in one sequencing reaction containing all four possible ddNTPs for each DNA molecule being sequenced. These fluorochromes are detected by fluorescence spectroscopy. Determining the fluorescence color of each band as it passes by the detector produces the nucleotide sequence of the template strand

Question 61

Next generation sequencing

Answer 61

A group of automated techniques used for rapid DNA sequencing. It has revolutionized molecular genetics because sequencers are low cost and can quickly generate sequences of thousands or millions of short fragments

Question 62

454 sequencing (pyrosequencing)

Answer 62

A DNA sample is fragmented into 400-600 base pair single strand fragments, which are modified with the addition of DNA adapters to both ends of each fragment. Each DNA fragment is immobilized on a bead and amplified by PCR. Primers are designed to anneal to the adapters, which creates a bead containing many copies of that DNA fragment. Each bead is put into a separate well with sequencing enzymes. and then 4 nucleotides are added to the well. When each nucleotide is incorporated, pyrophosphate is released as a byproduct of polymerization and releases a small flash of light. The light is recorded by a detector. This provides the order of nucleotides incorporated as a new strand of DNA is made

Question 63

Genomics

Answer 63

The study and comparison of entire genome, including the complete set of genes and their nucleotide sequence and organization

Question 64

Transcriptomics

Answer 64

The science of the entire collection of mRNA molecules produced by cells. Scientists can compare gene expression patterns between infected and uninfected host cells

Question 65

Metagenomics and metatranscriptomics

Answer 65

When genomics and transcriptomics are applied to entire microbial communities. It allows researchers to study genes and gene expression from a collection of multiple species

Question 66

Pharmacogenomics

Answer 66

Also called toxicogenomics. Involves evaluating the effectiveness and safety of drugs on the basis of information from an individual’s genomic sequences. Changes in gene expression in the presence of a drug can sometimes be an early indicator of the potential for toxic effects

Question 67

Proteomics

Answer 67

An extension of genomics that allows scientists to study all of the proteins in an organism (the proteome). All cells have the same DNA, but cells in various tissues produce different sets of proteins- the proteome is dynamic within the organism. It can be used to study which proteins are expressed under various conditions in a single cell type

Question 68

Biomarkers

Answer 68

Identifying proteins whose expression is affected by the disease process

Question 69

Bioinformatics

Answer 69

The collection, compilation, and analysis of large amounts of genetic information. It can be used for clues to treating diseases and understanding the workings of cell

Question 70

Reverse genetics

Answer 70

Used to determine the function of specific genes. It starts with a specific DNA sequence and attempts to determine which phenotype it produces

Question 71

Recombinant DNA pharmaceuticals

Answer 71

Pharmaceuticals produced through genetic engineering. Antibiotics can be produced through the targeted inactivation genes and the novel combination of antibiotic synthesis genes in Streptomyces bacteria. It is also used to make insulin and human growth hormone

Question 72

Antisense RNA

Answer 72

Molecules that are complementary to regions of specific mRNA molecules found in both prokaryotic and eukaryotic cells

Question 73

RNA interference (RNAi)

Answer 73

A natural regulatory mechanism where mRNA molecules are prevented from guiding the synthesis of proteins. Interference occurs when single stranded antisense RNA molecules base pair to a specific gene. Cells can use this to protect themselves from viral invasion

Question 74

RNA interference technology

Answer 74

Uses small interfering RNAs (siRNAs), which are complementary to the mRNA transcript of a specific gene of interest. These double stranded RNAs are bound to an endonuclease (DICER) that cleaves the RNA into short molecules. A protein complex binds to mRNA and inhibits translation

Question 75

Gene therapy

Answer 75

A clinical application of genetic engineering that might eventually be a cure for many genetic diseases

Question 76

How does gene therapy work?

Answer 76

It attempts to correct genetic abnormalities by introducing a nonmutated, functional gene into the patient’s genome. The healthy gene codes for a protein that a patient wouldn’t be able to produce otherwise. Viral vectors like the adenovirus can be used to introduce the new gene- the viral genome is removed and replaced with the desired gene