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What kind of enzyme was used to mix together human DNA and bacterial DNA to make the human gene for insulin? What is then used to mix together these cut up pieces?
- Restriction enzymes
- DNA ligase
What is a vector and what is an example?
- Anything that can carry DNA into a cell, which is the goal of making a recombinant DNA.
- Examples are plasmids and viruses.
Define a genome. What is a genomic library?
- Genome: all the DNA of an organism
- Genomic library: A place where genes are stored. An example of this is a population of bacteria or viruses that have been genetically engineered to contain copies of the genes that are being stored.
What does PCR stand for and what is its main purpose?
Polymerase Chain Reaction: it is used to make many copies of one specific piece of genome.
How is PCR started using primers?
- Two special primers, or two single-stranded DNA primers that are complementary to the DNA are made on either side of the target DNA.
- Then DNA polymerase will begin making new DNA, starting at these primers.
What is the type of DNA polymerase that is often used in PCR? Why is it used in PCR?
- Taq polymerase: it is isolated from the thermophilic bacterium, Thermophilus aquaticus.
- It is used because it is stable at the high temps necessary to run the reactions in a machine called a thermocycler.
What is the thermocycler?
A machine that is used to heat and seperate the strands of double-stranded DNA during PCR process.
Explain the process of the Polymerase chain reactioni (PCR)
- 1. Primers are made that are complementary to DNA on either side of the gene of interest.
- 2. In a tube, you combine: DNA to be copied, lots of primers, nucleotides, and TaQ polymerase (DNA polymerase)
- 3. The tube is heated in the thermocycler for 1 min at 95 degrees celcius. This is to seperate the double stranded DNA.
- 4. Thermocycler is cooled to 65 degrees for 1 min, allowing hydrogen bonds to form between DNA strands, and the numerous amounts of primers.
- 5. Temperature is raised to 75 degree celcius, the optimal temp for TaQ. TaQ makes new DNA that is complementary to the original DNA strand beginning at the 3' primers.
- 6. 1 strand of DNA now turns into 2, and this process is repeated until a billion copies of a single specific piece of DNA is made.
Why can't human DNA be directly inserted into bacterial DNA to make recombinants?
- In eukaryotes, mRNA have exons (broken up sections in the gene) that are seperated by introns.
- These introns are removed by enzymes after transcription and before translation, but most bacterial genes do not contain introns.
- If these introns cannot be broken up by bacterial cells, the protein produced would not be the normal human protein.
Fill in the blank:
In eukaryotes, the code for protein synthesis (mRNA) is boroken up into sections called __a__. The __a__ are seperated by pieces of DNA called __b__ that will not be used to make the protein.
What is cDNA and how is it produced?
(Include: an intron free mRNA template, use of viral enzyme, going from single stranded cDNA to double strand)
- Complementary DNA: Copy of human DNA introduced to bacterial genes without the introns to interrupt protein synthesis.
- 1. A template is made of human mRNA that already had its introns removed.
- (**Bacterial genes do not have introns, and will not produce human proteins if these introns aren't removed**)
- 2. A viral enzyme is then used, called reverse transcriptase, that makes DNA from RNA. Here, it produces a single strand of cDNA.
- 3. Other enzymes are then used to remove the mRNA, which is then followed up with DNA polymerase to synthesize a second strand of DNA complimentary to the first strand of cDNA.
- 3. A double stranded cDNA is produced, which is all the necessary coding information for the building of human proteins using bacteria.
What is happening at each step during this process of making cDNA?
- A. Starting with human DNA, mRNAs are made from gene of interest with the introns already removed.
- B. Reverse transcriptase is used (viral enzymes) to create a single strand of cDNA. This works by the viral enzyme creating DNA from RNA.
- C. More enzymes are then used to remove the mRNA, and DNA polymerase is used to turn it into a double stranded cDNA.
- D. The newly made cDNA is inserted into a plasmid.
- E. From here, the cloned cDNA (plasmid) is taken up by the target bacteria, and the bacteria can create human proteins.
What are DNA probes used for? How?
- These are used to identify which pieces of DNA, or which clones, contain a particular gene.
- 1. First, the DNA strands are heated or chemically seperatedinto single strands.
- 2. The probe is marked with an identifiable tag, which is then allowed to mix with the single stranded DNA.
- 3. The probe will then stick to the complementary sequence, revealing the location of the gene of interest.
This term means the process of killing or removing all viable organisms
This term means any process that removes things that cause disease, but doesn't remove ALL organisms.
What is sepsis?
When pathogenic organisms are present
What is asepsis?
When no pathogenic organisms are present.
What are antiseptics?
- A particular group of disinfectants – used to reduce the number of viable organisms in the skin. act differentially on organism and host tissue
- Examples are:
- 1. Germicidechemical agent capable of killing microbes
- 2. Sporicide a germicide capable of killing bacterial spores
What are bacteriostatic and bacteriocidal agents? Give examples of how each work.
- They are both antibiotics.
- Bacteriostatic agent: works by inhibiting the growth of bacteria.
- Example of this is tetracyclin. It disrupts bacterial ribosomes, which stops the bacteria from making proteins, and without protein production, cell replication doesn't work.
- Bacteriocidal agents: Kills bacteria directly
- An example of this is polymyxin B, which injures the plasma membranes of bacteria, allowing the cell contents to leak out.
T or F: Heat is the most effective and rapid method of sterilization and disinfection
How does sterilization by most heat work?
Acts by denaturation and coagulation of protein, breakage of DNA strands, and loss of functional integrity of cell membrane.
What are the different methods of moist heat sterilization?
- Sterlization at 100 degrees celcius:
- 1. boiling
- 2. steaming
- Sterilization above 100 degrees celcius:
- 3. Autoclaving - one of the most common methods. Done by steam under pressure, like a pressure cooker. It kills spores.
- Below 100 degrees celcius:
- 4. Pasteurization
How do you check the efficiency of autoclaving?
- Spores are used to see if autoclave is strong enough to kill it.
- Spores withstand 121 degrees celcius heat for up to 12 mins.
What is a con of using moist heat (autoclave)? (3 things)
- Has to be liquid or corrosioin will occur my moiust heat
- No petri plates, it can't stand heat
- No powder
How much temp for dry heat?
Dry powder: 160 degrees celcius
T or F: pasteurizing kills spores
What is pasteurizing mainly used for?
Wine and beer
What do hospitals use dry heat for? What temp and for how long?
Sterilizationi of glassware and instruments: 170 degrees for 2 hours
What are the two types of radiation used to kill bacteria?
- Ionizing radiation
- Ultraviolet radiation
This type of radiation causes thymine dimers in bacterial DNA, that disrupts protein synthesis and kills the cell.
This type of radition is used to sterilize drugs and disposable medical supplies such as plstic syringes and gloves
This tech uses dna mutation, and can be used to put in a specific place