Chapter 8 Recombinant DNA Technology

Why do scientists use mutations?

-Change phenotypes by changing genome -Select for and culture cells w/beneficial characteristics (more potent penicillin ) -Can isolate mutated genes from the rest of the organism (easier than dealing with whole gene/cell/organism)

What is the difference btw a gene library & a cDNA library?

-Gene library contains whole genome of of an organism -cDNA library only contains expressed genes of organism that the mRNA has copied

What are synthetic nucleic acids? Describe the 4 uses of synthetic nucleic acids?

-Molecules of DNA and RNA produced in cell-free solutions (in vitro) 1. explains the genetic code: to understand genetic code & amino acid sequence of a protein 2. Create genes for specific proteins: (e.g. synthesize a gene for human insulin) 3. Synthesize DNA & RNA probes to locate specific sequences of nucleotides: using synthetic nucleic acids and radioactive chemicals (probes) for detecting genetic sequences 4. Synthesizing antisense nucleic acid molecules: antisense nucleic acid molecules control genetic diseases

What is a DNA microarray? Describe the manufacture of DNA microarrays?

-multiple copies of a single-stranded DNA with known sequences on a substrate (i.e. glass slide, silicon chip, nylon membrane) -way of seeing if an mRNA molecule will be transcribed from gene to cell -fluorescently labeled DNA washed over the microarray binds to complementary strands

What is a genetic vector? What are the 4 useful properties that vectors share?

-nucleic acid molecules such as viral genomes, transposons, & plasmids. Vectors must: 1. be small enough to manipulate in a lab 2. able to survive inside cells: plasmids make good vectors b/c they are more stable than are linear fragments of DNA, which are typically degraded by enzymes 3. contain a genetic marker: help identify the specific gene of interest, can be phenotypic markers (i.e. gene that code for antibiotic resistance) or code for enzymes that metabolize a unique nutrients, or radioactive or fluorescent labels. 4. ensure genetic expression: by providing promoters

Describe the process of southern blotting? What are its uses?

-southern blot localizes specific DNA sequences (northern blot used to detect RNA molecules) on a stable membrane -uses gel electrophoresis to transfer DNA from agarose gels to nitrocellulose membranes Uses: 1. fingerprinting 2.diagnosing infectious diseases (e.g. can detect the presence of genetic sequences unique to hepatitis B in a blood sample of an infected patient) 3. to demonstrate the incidence & prevalence in an environmental sample of archaea, bacteria, and viruses, particularly those that cannot be cultured

What are the three main goals of recombinant DNA technology? Give examples of each.

1) eliminate undesirable phenotypic traits in humans, animals, plants, and microbes (e.g. pest-resistant plants, cures for genetic disorders) 2) to combine beneficial traits of two or more organisms to create valuable new organisms 3) to create organisms that can synthesize products that humans need (e.g., produce vaccines, antibiotics, hormones, or enzymes)

List & explain 3 artificial techniques for introducing DNA into cells?

1. Electroporation: "shock shell" an electrical current is applied to a cell to permit it to take up DNA. -Cell become recombinant or transgenic cell -for fungi and algae, you have to enzymatically remove cell wall to make it into a protoplast 2. Protoplast fusion: cytoplasmic membranes may fuse to form a single cell that contains genome of both cells. Polyethylene glycol (antifreeze) increases rate of fusion. 3. 2 types of Injection are used w/ larger eukaryotic cells: a. gene gun: powered by a blank .22 caliber cartridge or compressed gas to fire tiny gold beads coated w/DNA into target cell b. microinjection: inserts DNA into a target cell via a glass micropipette -injection can be used on intact tissues such as in plant seeds

What are the 5 tools required for recombinant DNA technology? Give an example and describe the importance of each tool.

1. Mutagens: physical & chemical agents that produce mutations. Use to create changes in microbes' genomes so that the microbes phenotypes are changed 2. Reverse Transcriptase: uses RNA template to transcribe a molecule of DNA (i.e. cDNA--> RNA) 3. Synthetic nucleic acids: used as DNA probes, primers, and antisense RNAs 4. Restriction enzymes: Bacterial enzymes that cut DNA molecules only at restriction sites (usually palindromes) -Normally protect bacteria from phage by chopping up its DNA 5. Vectors: Nucleic acid molecules that deliver a gene into a cell.

Explain how a cDNA is formed: (3 steps)

1. mRNA is extracted from cells, and reverse transcriptase is extracted from retroviruses. These are used in vitro to turn the mRNA into single stranded DNA transcripts. 2. The 3' end of the mRNA has a stretch of Adenine ribonucleotides, called a poly-A tail. A short strand of thymine deoxyribonucleotides can be added here as a primer for reverse transcriptase. 3. Reverse transcriptase transcribes the RNA to a single stranded DNA molecule. Then a 2nd DNA strand, complementary to the first, is synthesized by DNA polymerase. The resulting double stranded DNA is called a complementary DNA (cDNA) and contains only exons.

What is a gene library and explain its beneficence?

A gene library is the population of cells or phages that together contain all of the genetic material of interest.

What are the uses of DNA microarrays?

DNA microarrays allow scientists to study many genes and their expression, at once. 1. Monitoring gene expression: monitor which genes are transcribing at a particular time by making fluorescently labeled cDNA from mRNA in the cell 2. Diagnosing infection: reveals the presence of pathogens 3. Identifying organisms in an environmental sample: monitor the presence/absence of microbes in an environment by using microarrays of DNA from the organisms

Describe the process & use of gel electrophoresis.

Gel electrophoresis is a technique that involves separating molecules based on their electrical charge, size, and shape, which can be used to isolate fragments of DNA molecules that can be then inserted into vectors, multiplied by PCR, or preserved in a gene library. -smaller DNA fragments move faster & farther than larger ones.

Describe nucleotide sequencing and its usefulness.

Genomics: the sequencing and analysis of nucleotide bases of genomes. Automated. Uses cDNA, nucleotides tagged with 4 different dyes -useful to develop novel drugs and more effective therapies and vaccines

Describe genetic mapping and its usefulness.

Involves locating genes on a nucleic acid molecule Provides useful facts concerning metabolism, growth characteristics, and relatedness to others

Describe the purpose and process of polymerase chain reaction (PCR).

Multiplying DNA in vitro Critical to amplify DNA in variety of situations: 1. Denaturization: high temps (~94 degrees) melts DNA, break hydrogen bonds 2. Priming: cool lower temp. to ~60 degrees to allow primers to bind 3. Extension: raise temp (72) to make reaction faster to produce more DNA, copying with DNA polymerase 4. Repeat: each cycle produces double amount of DNA

Describe the actions of restriction enzymes and the 2 types.

Restriction enzymes recognize & cut both strands of DNA molecule at a specific restriction site (usually palindromic) and produce either: a. sticky ends: staggered cuts, which only bind to complementary fragments produced by same restriction enzyme b. blunt ends: non-sticky and non-specific, which enables DNA cut by differ restriction enzymes to be combined easily.

What is recombinant DNA technology? Describe the process.

Technique that allows DNA to be combined from different sources; also called gene or DNA splicing. Recombinant DNA is an important technique for many gene-cloning applications. 1) Isolate plasmid: probe is used to isolate the gene of interest 2) Enzymatically cleave DNA into fragments: restriction enzyme that can locate and cut the gene from the DNA segment, cuts an opening in the recipient DNA (usually a plasmid) where the donor DNA can be attached 3) Isolate fragment w/ gene of interest: DNA ligase attaches donor and recipient DNA, forming a recombinant DNA molecule 4) Insert gene into plasmid: recombinant DNA molecule is inserted into host cell 5) Insert plasmid & gene into bacterium 6) Culture bacteria

What is biotechnology?

The use of microorganismsto make practical products

What is a plasmid?

loops of DNA that are separate from the bacterial chromosome and that replicate on their own within the cell

How are DNA probes used to identify recombinant cells?

probes bind exclusively to their complementary nucleotide sequences and have either radioactive or fluorescent markers. Fluoroscent/radioactive cells are isolated and cultured, which aids in identifying the specific location of the genes of interest

How do researchers develop vectors? Give an example.

researchers are developing vectors from adenoviruses, poxviruses, and a genetically modified form of HIV. HIV might make an excellent vector b/c HIV inserts itself into human chromosomes.