Biotechnology and Molecular Biology Flashcards Preview

BIO 233 > Biotechnology and Molecular Biology > Flashcards

Flashcards in Biotechnology and Molecular Biology Deck (61):
1

Biotechnology

Biotechnology is the exploration of biological processes for industrial and other purposes, especially the genetic manipulation of microogranisms for the production of antibiotics and hormones 

2

Molecular biology

Molecular biology is the study of biological organisms at the molecular level

3

  • Use of biological systems to make desired end products
  • Can use whole organisms or components of an organism (enzymes)
  • Does not require genetically engineered organisms 

Biotechnology 

4

  • Intentional modification of genomes for practical purposes
  • Deliberate alteration of genotype to change phenotype
  • Created using biotechnology and molecular biology tools 

Genetic Engineering 

5

  • The study of organisms at the molecular level
  • Includes genetics, biochemistry, microbiology, biotechnology, and genetic engineering\
  • Relies on the use of DNA and RNA and their associated enzymes

Molecular Biology 

6

What are the tools of molecular biology?

Tools of Molecular Biology

  1. PCR
  2. Gel Electrophoresis
  3. DNA microarrays
  4. Recombinant DNA
  5. Blots
  6. CRISPR System/ Cas 9

7

PCR stands for?

Polymerase Chain Reaction

8

In vitro DNA replication

  • Can "amplify" small amounts of DNA into lots of DNA
  • Can detect specific genes
  • Can be used to clone or copy specific sequences
  • Used in medicine, forensics, empirical research

PCR

9

  • Uses automated equipment (thermocycler) to run through several replication cycles
  • Each replication cycle doubles the amount of DNA produced
  • Technology possible due to Taq polymerase, a heat stable DNA Polymerase

PCR

10

  • Consists of three steps
  • Length of and the number of cyles varies
  • Reaction mixture contains target DNA, A,T,C,G, primers, enzymes, and buffers

PCR Cycle

11

What are the three steps of the PCR Cycle

PCR Cycle

  1. Denaturation
  2. Priming (annealing)
  3. Extension 

12

  • Heat is used to separate double strands of the DNA molecule so existing strands can be used as templates
  • Hydrogen bonds break at ~95 degrees celsius
  • Mimics activity of Helicase

Denaturation 

  • First step in PCR cycle

13

14

  • DNA primers from the hydrogen bonds to the target sequence
  • The primers are short (~25 bp) of synthetic pieces of DNA that are complementary to the target sequence of DNA
  • One primer is required for each template strand
    • Specific primers have complementary bases (correlates to specific genes)
  • Priming step usually around 55 to 65 degrees celsius
  • Mimics activity of primase

Priming (Annealing)

  • Second step of PCR Cycle

15

  • Taq polymerase (DNA polyermase) replicates template DNA
  • Named after the source organism Thermus aquaticus
  • Temperature usually at 72 degrees Celsius
  • Can copy 1000 bp in 10 seconds (100 bp/sec)
  • Times vary based on length of piece of DNA being copied 

Extension

  • Third and final step of PCR Cycle

16

  • Technique used to analyze pieces of nucleic acids (DNA or RNA) that uses a gel matrix and electricity
  • Gel is made from agarose, the purified sugar from agar
  • DNA and RNA are negatively charged (phosphate groups), and move towards a positive charge
  • When set up, nucleic acids travel through gel, and are separated by size

Gel Electrophoresis 

17

Q image thumb

PCR

18

Q image thumb

Denaturation

  • First step of PCR Cycle
  • ~95 degrees celsius to break hydrogen bonds
  • Mimics
  • Helixase

19

Q image thumb

Priming (Annealing)

  • Second step of PCR Cycle
  • 55 to 65 degrees Celsius
  • Mimics primase

 

20

Q image thumb

Extension

  • Third and final step of PRC Cycle
  • Temperature 72 degrees Celsius
  • Mimics DNA polymerase
  • Uses Taq polymerase

21

Q image thumb

Gel Electrophoresis 

22

Q image thumb

Gel Electrophoresis 

23

  • Smaller DNA/RNA fragments migrate faster and therefore farther from the starting point
  • The size of the DNA/RNA fragments can be determined by comparing strands
  • Is a routine procedure used to visualize PCR products, samples of mRNA from cells, quality of isolated DNA

Gel Electrophoresis 

24

  • Process brings with gel electrophoresis and tranfers DNA/RNA fragments to a fancy piece of paper

Blots

25

What is a problem of the blot technique?

The blot technique can only test for one gene or mRNA at a time (very time consuming)

26

What is the type of blot used to detect a specific gene in a mixed sample of DNA pieces?

Southern Blot

 

27

What is the type of blot used to detect a specific piece of mRNA in a mixed sample of RNA?

Northern blot

28

Why is the Northern blot considered more useful than the Southern blot?

  • DNA replicating involves RNA
  • Northern blots are useful in understanding how a gene is expressed in relation to environmental factors 

29

What does both the Northern and Southern blot require in terms of tools?

  • Use of a probe (primer)
  • A synthetic piece of DNA designed to complement a specific nucleic acid target 

30

  1. Pieces of nucleic acid first separated on a gel
  2. Nucleic acids from gel are then transferred to a membrane, often nylon, that is easier to handle- this is the blot
  3. A radioactive or fluorescently active probe is exposed to the blot
  4. The probe will stick only to nucleic acid fragments of interest

Procedure for Northern & Southern Blots 

31

Q image thumb

(Southern) Blot 

32

Q image thumb

Gel and Blot

33

  • Technique often used to study the simultaneous expression (transcription) of ALL of the genes in an organism under specific conditions
  • Based on blot technology
  • Unlike blot, nucleic acid samples are added to probes, not probes to samples 

DNA Microarray 

34

  1. Single stranded DNA probes are affixed to glass slide (microscope slide)
  2. To study gene expression, mRNA from the cell is collected
  3. Reverse transcriptase is used to convert mRNA to cDNA (complementary DNA)
  4. cDNA is labeled with fluorescent compounds
  5. cDNA solution is washed over slide, analyzed by computer

Procedure for DNA Microarrays

35

cDNA

Complementary DNA

36

Q image thumb

DNA Microarray

37

Q image thumb

DNA Microarrays

38

In a DNA microarray what do the colors indicate in terms of the genes and conditions before hybridization?

  • Red = reference cDNA (control)
  • Green = experimental cDNA

39

What is hybridization?

The process in which the cDNA molecule binds to the DNA probe is called hybridization

40

In a DNA microarray what do the colors indicate in terms of the genes and conditions after hybridization?

  • Equal gene expression in both samples = yellow dot
  • Higher gene expression in experimental than reference = red
  • Lower gene expression in experimental than reference= green

41

  • DNA made from combining DNA from one or more sources into novel DNA molecules
    • Splices genes from same organism
    • Splices genes from different organisms 
  • Then placed in host cell for gene expression 

Recombinant DNA

42

Q image thumb

Recombinant DNA

43

  • Bacterial enzymes that cut large molecules of DNA into smaller pieces
  • Cuts DNA molecules only at specific sequences called restriction sites
  • Leaves overhangs for hydrogen bonding
  • Named for organism they are found in
    • EcoRI, SmaI, HindIII

Restriction Enzymes

44

  • Restriction sites are palindromic sequences of DNA
  • Each restriction enzyme recognizes a single, specific recognition site
    • EcoRI-GAATTC/____________
    • HindIII- AGCTT/____________
    • SmaI CCCGGG/____________

Restricition Enzymes

  • EcoRI- GAATTC/CTTAAG
  • HindIII- AGCTT/ TTCGA
  • SmaI- CCCGGG/GGGCCC

45

Q image thumb

Restriction Enzymes 

46

Q image thumb

Restriction Enzymes 

47

  • Insert recombinant vector into host organism
    • Transformation
    • Electroporation 

Genetically Modified Organisms/ Bacteria 

48

  • Bacterical cells are exposed to a powerful electrical field
  • Voltages as high as 2.5 kV
  • Creates transient openings in cell wall and membrane
  • Allows DNA to diffuse in 
  • Very high efficiency transformation using very small amounts of DNA

Recombinant DNA Electroporation 

49

Q image thumb

Recombinant DNA Electroporation 

  • Cells are exposed to powerful electrical field
  • Voltages as high as 2.5 kV
  • Creates transient openings in cell wall and membrane
  • Allows DNA to diffuse in
  • Very high efficiency tranformation using very small amounts of DNA 

50

  • Clustered Regularly Interspaced Short Palindromic Repeats 

Q image thumb

CRISPR/Cas System 

  • Found in many Bacteria and Archaea
  • Provides immunity to bacteriophage and plasmids
    • Spacers contain DNA samples from plasmids and viruses that bacterium is previously exposed to
      • Type of acquired immunity
    • Cas proteins are often nuclease, and serve to cut target DNA sequences 

51

  • Found in many Bacteria and Archaea
  • Provides immunity to bacteriophage and plasmids
    • Spacers contain DNA samples from plasmids and viruses that bacterium is previously exposed to
      • Type of acquired immunity
    • Cas proteins are often nucleases, and serve to cut target DNA sequences

CRISPR/Cas System 

52

Q image thumb

CRISPR/Cas System

  • Found in many Bacteria and Archaea
  • Provides immunity to bacteriophage and plasmids
    • Spaces contain DNA samples from plasmids and viruses that bacterium is previously exposed to 
      • Type of acquired immunity
    • Cas proteins are often nucleases, and serve to cut target DNA sequences 

53

  • In bacteria, spaces regions encode for crRNA- crisprRNA
  • crRNA serves as a guide to direct ____protein to target DNA
  • In biotech applications, an artifical crRNA is created (gRNA, guide RNA) and this directs specific targeting for directed DNA cutting 

Cas9

  • Crispr associated protein 9

54

What are some applications for CRISPR/Cas? 

  1. Gene knockouts for inactive genes
  2. DNA insertion
  3. Antiviral therapy
  4. Gene therapy 

55

What are some applications for biotech?

Drugs/Hormones

  • Human insulin derived from gene in yeast
    • Genetically modified organism 

56

What are some applications in biotech?

Vaccines?

  • Subunit vaccines

57

What are some applications in biotech?

Genetic Screening

  • DNA microarrays are used to screen individuals for inherited diseases caused by mutations

58

What are some applications in biotech?

Infection Diagnosis

  • PCR
  • Patient specimens can be examined for presence of gene sequences unique to certain pathogens 

59

What are some applications in biotech?

Industrial Microbiology

  • Recombinant bacteria to produce biofuels 

60

What are some applicatins in biotech?

Genomics

  • Genomics is the study of the entire genome of an organism
  • Hundreds of bacterial species' genomes are sequenced
  • Cows, bees, mice, rats, and others are completely sequenced
  • Human genome project 

61

What are some applications of biotech?

Microbiomes

  • Microbiomes is the analysis of microbiological populations of an organism
  • Based on DNA sequencing of mixed samples
  • Technique used to recognize the bacteria in the gut affects the metabolism of humans
  • Similiar techniques used to study other microbial populations