study guide micro exam 2

1. What are transposons? What do they do? 

Transposons, or jumping genes, are DNA sequences that move within a genome, discovered by Barbara McClintock. They can cause mutations, alter gene expression, drive evolution, or contribute to diseases like cancer. 

Types: 

1. Retrotransposons (Class I) – Copy-paste via RNA. 

2. DNA Transposons (Class II) – Cut-paste using transposase. 

They make up about 45% of the human genome and play a key role in genetic diversity. 

2. Understand impact of frameshift, deletion, insertion mutations. 

Impact of Different Mutations: 

1. Frameshift Mutation (Caused by insertions or deletions): 

1. Shifts the reading frame of the genetic code. 

2. This leads to completely different amino acids being translated. 

3. Often results in nonfunctional proteins or premature stop codons. 

4. Example: Inserting/deleting a nucleotide in a gene like CFTR can cause cystic fibrosis. 

2. Deletion Mutation: 

1. Removes one or more nucleotides from DNA. 

2. If in multiples of three, it may remove an amino acid but keep the reading frame. 

3. If not in multiples of three, it causes a frameshift mutation. 

4. Example: Duchenne muscular dystrophy (DMD) is caused by deletions in the dystrophin gene. 

3. Insertion Mutation: 

1. Adds extra nucleotides to DNA. 

2. Like deletions, if not in multiples of three, it leads to a frameshift. 

3. Can create nonfunctional or toxic proteins. 

4. Example: Huntington’s disease occurs due to excessive CAG repeats (trinucleotide insertion). 

3. What is the effect of intercalating agents 

Intercalating agents insert between DNA base pairs, causing mutations, replication errors, and DNA damage. They can lead to frameshift mutations, disrupt gene expression, and trigger cell death. Some are carcinogenic, while others serve as chemotherapy drugs. 

4. Describe the properties of mutagens; How do they bring about the change; What actually changes? 

Mutagens are agents that increase DNA mutations. They can be physical (radiation), chemical (alkylating agents), or biological (viruses, transposons). 

How They Cause Changes: 

• DNA damage (breaks, crosslinking). 

• Base modifications (mispairing, deamination). 

• Replication interference (frameshifts, strand breaks). 

What Changes? 

• Point mutations, insertions/deletions, chromosomal alterations → Can lead to genetic disorders, cancer, or evolution. 

5. What is the effect of base analogs in a gene sequence? 

Base analogs mimic DNA bases, causing mispaired substitutions and increasing mutation rates. They induce point mutations via tautomeric shifts, leading to genome instability. 

Examples: 

• 5-Bromouracil (5BU) – Pairs with guanine, causing A-T → G-C mutations. 

• 2-Aminopurine (2AP) – Pairs with cytosine, leading to transitions. 

These mutations can result in genetic disorders, cancer, or antibiotic resistance. 

6. What is the effect of Intercalating agents in a gene sequence 

Intercalating agents insert between DNA base pairs, distorting the helical structure. This leads to: 

• Frameshift mutations (insertions/deletions during replication). 

• Disrupted transcription & replication (polymerase errors). 

• DNA breaks & instability, potentially causing cancer. 

Examples: 

• Ethidium bromide, acridine orange – Induce frameshifts. 

• Doxorubicin – Used in chemotherapy to disrupt cancer cell replication. 

7. Describe the DNA repair mechanism. 

1. Describe mismatch repair  

Mismatch repair fixes errors made during DNA replication, like mispaired bases or small insertions/deletions. It identifies the error based on methylation marks (in bacteria) or other signals, removes the incorrect DNA segment, and fills in the gap using the correct strand as a template. 

2. What is the relationship between mutagens and antibiotics 

Mutagens are substances that increase mutation rates, while antibiotics are substances that inhibit bacterial growth or kill bacteria. Some antibiotics can act as mutagens by inducing genetic changes in bacteria, which may lead to antibiotic resistance. 

3. What is indirect selection? 

Indirect selection is a technique where mutants are selected based on their ability to grow or survive in conditions where non-mutants cannot. Auxotrophic mutants (which require a nutrient not needed by wild-type organisms) can be selected by growing them in a medium lacking that nutrient. 

4. What is replica plating?  

Replica plating is a method for identifying specific mutants. A colony of microorganisms is transferred to a new agar plate with different conditions to see which colonies grow under those conditions, helping researchers identify mutants(e.g., antibiotic-resistant strains). 

5. Describe the AMES test. 

The AMES test is a method for detecting mutagenic substances. It uses bacteria (often Salmonella) that have a mutation in a gene for histidine synthesis. If a substance is a mutagen, it will cause a reverse mutation that restores histidine production, allowing the bacteria to grow on a histidine-free medium. 

6. Define genetic recombination. 

Genetic recombination is the process where genetic material is exchanged between different DNA molecules. It can happen naturally (in sexual reproduction or during horizontal gene transfer) or artificially in labs. This leads to genetic diversity. 

7. Describe transformation, conjugation, and transduction. 

Transformation: The process where bacteria take up free DNA from the environment. This DNA can integrate into the bacterium's genome, leading to new traits (e.g., antibiotic resistance). 

Conjugation: The transfer of DNA between bacteria via direct contact. A plasmid (small circular DNA) or part of the bacterial chromosome can be transferred using a pili. 

Transduction: The process where viruses (bacteriophages) carry bacterial DNA from one cell to another. This can introduce new genes into the recipient bacterium. 

8. What is the role of plasmid in recombination/conjugation? 

In conjugation, plasmids are small, circular DNA molecules that can replicate independently of the bacterial chromosome. They can be transferred between bacteria, often carrying antibiotic resistance genes or other traits. During recombination, plasmids can integrate into the chromosome or transfer their genes to other cells, contributing to genetic diversity. 

9. What is the Crown gall tumor? 

Crown gall is a plant disease caused by the bacterium Agrobacterium tumefaciens. The bacteria transfer part of their DNA (the Ti plasmid) into the plant cells, causing the formation of tumors (galls) at the crown of roots or stems. 

10. What is the function of DNA Polymerase? 

DNA polymerase is an enzyme that synthesizes new DNA strands by adding nucleotides to a growing DNA chain during DNA replication. It also plays a role in DNA repair and proofreading to ensure accuracy in copying the genetic material. 

11: What is CRISPER? How does it workCRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a genetic editing tool derived from bacteria. It works by using guide RNA (crRNA) to target specific DNA sequences, and the Cas9 nuclease cuts the DNA at the targeted location, allowing for gene editing or insertion. 

12. How do selectable markers work in clone selection?Selectable markers are genes introduced into a cloning vector that confer a trait (e.g., antibiotic resistance). After introducing a plasmid into a host, only cells that have taken up the plasmid with the marker will grow in the presence of the selective agent, allowing researchers to identify successful clones. 

13. What is the PAN genome? 

The PAN genome refers to the total set of genes found in a species population, including both core genes (common to all strains) and accessory genes (present in some strains but not all). It highlights the genetic diversity within a species. 

14. What is the CAS nuclease crRNA complex (CRISPR)? 

The Cas9 nuclease-crRNA complex is the molecular machinery used in CRISPR gene editing. The crRNA guides the Cas9 nuclease to a specific DNA sequence, where Cas9 cuts the DNA, allowing for gene editing or modification. 

15. Define antisepsis, sterilization, disinfection, degerming, and sanitation. 

• Antisepsis: Prevention of infection by killing or inhibiting microorganisms on living tissues. 

• Sterilization: The process of completely removing or killing all microorganisms. 

• Disinfection: The removal or killing of most harmful microorganisms on non-living surfaces. 

• Degerming: The mechanical removal of microbes from a surface (e.g., hand washing). 

• Sanitation: Reducing microbial populations to safe levels on surfaces or objects. 

16. Define pasteurization. 

Pasteurization is the process of heating liquids (e.g., milk, juice) to a specific temperature for a set period to kill pathogenic microorganisms without affecting the taste or quality of the product. 

17. How do HAl's occur? 

Healthcare-associated infections (HAIs) occur when patients acquire infections during medical treatment or hospital stays, typically from contaminated medical instruments, healthcare workers, or the hospital environment. 

18. Why is boiling water used in disinfection? 

Boiling water kills many pathogens by exposing them to high temperatures (100°C) for several minutes, making it an effective method for disinfecting water, though it may not eliminate all types of microorganisms (e.g., bacterial spores). 

19. What is the purpose of an autoclave? What are the settings? 

An autoclave sterilizes equipment and materials by applying high-pressure steam (typically 121°C at 15 psi) for a set duration (e.g., 15-30 minutes). This method is highly effective at killing bacteria, viruses, and spores. 

20. What happens in the canning process? 

Canning involves sealing food in jars or cans and heating them to destroy microorganisms and prevent spoilage. The high temperature kills bacteria, yeast, and molds while creating a vacuum seal that prevents contamination after the container cools. 

21. How are X-rays, Gamma rays used in sterilization? 

X-rays and gamma rays are forms of ionizing radiation that damage the DNA of microorganisms, killing them or preventing them from replicating, making them effective for sterilizing medical equipment and some food products. 

22. Role of microwaves in sterilization? 

Microwaves use high-frequency radiation to generate heat in water molecules, which can kill or inactivate microorganisms. However, microwaves are not commonly used alone for sterilization as they don't consistently penetrate to all areas of the material being sterilized. 

23. What are chemical germicides? 

Chemical germicides are chemicals used to kill or inhibit the growth of microorganisms, including bacteria, viruses, and fungi. They include disinfectants, antiseptics, and sterilants. 

24. Action of alcohols?Alcohols (e.g., ethanol, isopropanol) denature proteins, disrupting cell membranes and leading to cell death. They are effective at killing most bacteria and viruses but not spores. 

25. Action of phenolics? 

Phenolics (e.g., phenol, cresols) disrupt cell membranes and denature proteins, making them effective in killing bacteria, fungi, and viruses. They are commonly used in disinfectants. 

26. Action of quaternary ammonium compounds? 

Quaternary ammonium compounds (quats) are surfactants that disrupt cell membranes, leading to leakage of cellular contents and microbial death. They are commonly used as disinfectants for surfaces. 

27. Action of silver sulfur diazine - what does it kill? 

Silver sulfadiazine is an antimicrobial compound used to treat burns. It kills bacteria and fungi by interfering with their cellular metabolism and protein synthesis. 

28. Action of dry heat in sterilization? 

Dry heat sterilization uses hot air (typically 160-180°C) for a prolonged period (1-2 hours). It works by oxidizing and denaturing proteins, killing microorganisms, including spores. 

29. How sterilization happens in microwave, pasteurization, filtration, irradiation and autoclaving? 

• Microwave sterilization: Kills microorganisms by generating heat in water molecules. 

• Pasteurization: Kills pathogens without changing the product significantly. 

• Filtration: Removes microorganisms from liquids or gases by physically trapping them. 

• Irradiation: Uses ionizing radiation to damage DNA and kill microorganisms. 

• Autoclaving: Uses high-pressure steam to sterilize equipment by denaturing proteins and killing all microorganisms. 

30. Action of nitrates and nitrates? 

Nitrates and nitrites are preservatives that inhibit bacterial growth, especially Clostridium botulinum (which causes botulism). They prevent the growth of certain bacteria by disrupting their metabolism. 

31. Action of chlorine? Chlorine is a powerful disinfectant that kills microorganisms by oxidizing their cell components (proteins, lipids, and DNA). It is widely used in water treatment and disinfection. 

32. What is a restriction enzyme?A restriction enzyme (also known as a restriction endonuclease) is a protein that cuts DNA at specific sequences. They recognize specific short DNA sequences and cleave the DNA, enabling processes like cloning and genetic modification. 

33. Describe the purpose and process of gel electrophoresis. Gel electrophoresis separates DNA, RNA, or proteins based on size and charge. 

a. Cathode/Anode 

• Cathode is the negative side, where DNA (negatively charged) is loaded. 

• Anode is the positive side, where the DNA moves towards. 

b. DNA is negatively charged due to phosphate backbone and is attracted to the positive pole. 

• DNA is negatively charged: Due to the phosphate backbone, DNA migrates toward the positive pole. 

c. Separate by size and charge 

Separate by size and charge: DNA fragments separate through the gel matrix based on size; smaller fragments travel faster. 

d. smallest pieces travel faster: Smaller DNA fragments move farther through the gel in a given time. 

e. Larger pieces stay closer to wells 

Larger pieces stay closer to wells: Larger DNA fragments face more resistance and travel slower. 

f. Lane 1: standard/size marker 

• Known DNA fragments are used to compare and estimate the size of unknown samples. 

34. What is a cloning vector? What are its major components? 

A cloning vector is a DNA molecule used to carry and introduce foreign DNA into a host cell for replication or expression. Major components: 

• Origin of replication: Ensures the vector replicates in the host. 

• Selectable marker gene: Allows identification of successful transformation (e.g., antibiotic resistance). 

• Multiple cloning site (MCS): A region with several restriction enzyme sites for inserting foreign DNA. 

35. Describe the purpose of a DNA microarray. 

A DNA microarray allows for simultaneous measurement of the expression of thousands of genes. It detects gene activity by hybridizing labeled cDNA with complementary DNA probes on a chip, allowing researchers to study gene expression patterns. 

36. Define genetic engineering. 

Genetic engineering is the process of manipulating DNA to introduce new genes or modify existing ones, often to produce desired traits or functions in organisms (e.g., transgenic crops, gene therapy). 

37. What is cDNA? RNA->DNA via reverse transcriptase (no introns) 

cDNA (complementary DNA) is synthesized from mRNA using reverse transcriptase. It represents only the exons of genes (without introns), making it useful for studying gene expression and producing recombinant proteins. 

38. Be able to describe the cloning process: 

a. PCR amplification 

• Use polymerase chain reaction (PCR) to amplify the target gene of interest. 

b. Restriction enzymes 

i. Cutting at matching insertion sites Restriction enzymes cut DNA at specific sites, preparing the gene for insertion into a vector. 

 ii. Ligation: The gene of interest is joined to the vector using ligase. 

Restriction enzymes cut DNA at specific sites, preparing the gene for insertion into a vector. 

c. Transformation with. competent cells 

i. Heat shock: introduces the recombinant plasmid into competent cells (e.g., E. coli). 

 ii. Ice/heat: After heat shock, cells are incubated on ice to help the plasmid integrate into the bacterial cells.  

d. Blue/White Screening 

i. Antibiotic: Selects for successful transformation - AmpR gene on vector (vector with gene and vector without gene).  

Selects for successful transformation. Only cells with the plasmid (which carries an antibiotic resistance gene) will grow on antibiotic plates. 

1. Growth: Successful transformation 

2. No growth: Unsuccessful transformation 

 ii. XGAl: Selects for successful ligation 

1. Blue colonies: No gene of interest (lacZ not interrupted; B-galactosidase is made; XGAL cleaves the B-galactosidase; blue color produced). 

2.White colonies: Gene of interest (lacZ interrupted; no B-galactosidase made; XGAL not cleaved; no blue color) 

39. Know the steps and stages of PCR 

a. Denaturation 

Heat DNA to 95°C to separate the double strands. 

b. Annealing 

Cool to 50-65°C to allow primers to bind to target sequences. 

c. Extension 

Heat to 72°C for Taq polymerase to synthesize new DNA strands. 

d. Enzymes used/purpose 

Taq polymerase (heat-stable enzyme) synthesizes new DNA. 

e. Temperatures 

• Denaturation: 95°C 

• Annealing: 50-65°C 

• Extension: 72°C (optimal temperature for Taq polymerase). 

40. CAS-9 role in the CRISPER 

Cas9 is a nuclease enzyme used in CRISPR gene editing to make precise cuts in DNA. It is guided to a specific DNA sequence by gRNA to facilitate targeted gene editing. 

41. gRNA in CRISPR 

gRNA (guide RNA) is a synthetic RNA molecule that guides Cas9 to the specific location in the genome by matching its sequence with the target DNA. 

42. What is a DNA library? 

A DNA library is a collection of cloned DNA fragments from an organism, representing its entire genome (genomic library) or expressed genes (cDNA library). These libraries are used for research and gene discovery. 

43. What are short tandem repeats? 

Short tandem repeats (STRs) are regions in DNA where short nucleotide sequences (2-6 base pairs) are repeated multiple times. They are used in forensic analysis and genetic profiling due to their high variability between individuals. 

44. E. coli- outer membrane (LPS) could be toxic to humans. 

The outer membrane of E. coli contains lipopolysaccharides (LPS), which can be toxic to humans, triggering an immune response and causing sepsis in some cases. 

45. DNA vs cDNA 

• DNA: Contains both introns and exons, and represents the full genetic code of an organism. 

• cDNA: Synthesized from mRNA and contains only the exons, used to study gene expression or produce proteins. 

46. Function of a probe in FISH - fluorescent in situ hybridization 

A probe in FISH is a labeled DNA or RNA fragment that binds to a complementary sequence in a sample. It allows the detection of specific genes or chromosomal regions under a microscope using fluorescent tags.