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Bio 120 Lab > Lab 7 > Flashcards

Lab 7 Flashcards

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Biology 101 flashcards
1
Q

Biotechnology

A

The use of organisms or their components (DNA, protein) to make or modify products useful to humans

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2
Q

Traditional Biotechnology

A

Has been utilized for centuries and includes selective plant and animal breeding and the use of yeast in fermentation to produce cheese, bread, beer and wine

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3
Q

Modern Biotechnology

A

Occurred in the 1970s and refers to the manipulation of DNA in vitro (in glass)
- Allows the alteration of specific DNA sequences in genes as well as the transfer of genes between organisms

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4
Q

What has Biotechnology enabled?

A

Advances in forensics, medicine, agriculture, pollution control, evolutionary biology

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5
Q

DNA:

1) It is a ___________ molecule
2) Each strand is composed of a series of _______
3) Each nucleotide is composed of three parts:
i) A _________ (one of adenine, cytosine, guanine, or thymine)
ii) A ______ (deoxyribose)
iii) A _________ (_______ charged)
4) Within each of the two strands of DNA, nucleotides are joined by ____________________
5) Between two strands of DNA, __________ are formed between ________________ (adenine pairs with _______, cytosine with __________)

A

1) It is a large double-stranded molecule
2) Each strand is composed of a series of nucleotides
3) Each nucleotide is composed of three parts:
i) Nitrogenous base (one of adenine, cytosine, guanine, or thymine)
ii) A sugar (deoxyribose)
iii) A phosphate group (negatively charged)
4) Within each of the two strands of DNA, nucleotides are joined by covalent phosphodiester bonds
5) Between two strands of DNA, hydrogen bonds are formed between complementary nitrogenous bases (adenine pairs with thymine, cytosine with guanine)

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6
Q

Central dogma

A

The flow of information from DNA to RNA to protein

- RNA makes polypeptides into amino acids

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7
Q

Biological samples collected for DNA analysis must contain _____ cells
EX?

A

Nucleated cells

EX. bodily fluids, hair, skin, organs, bones

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8
Q

What typed of biological samples are most commonly obtained from suspects for analysis?

A

Blood, hair, buccal (cheek) cells

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9
Q

What are the steps in processing and analyzing DNA collected from a crime scene?

A

Collection of Biological evidence –>
Step 1: DNA Extraction –>
Step 2: Polymerase Chain Reaction (PCR) –>
Step 3: Restriction Fragment Analysis –>
- Restriction Digest
- Gel Electrophoresis
Step 4: Interpreting the Results

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10
Q

Step 1: DNA Extraction

Define Extraction

A
  • Extraction of DNA from the nuclei of cells
  • Extraction is accomplished by chemically lysing (breaking open) the cells and organelles to liberate the DNA, which is then chemically precipitated out of solution
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11
Q

What are the 8 steps of Extraction from our cheek cells? And why do we do them?

A

1) Swish around 1 ml of Gatorade - Collects intact cheek cells - the saline solution is isotonic(equal movt of water into/out of the cell)
2) Spit it out into cup
3) Pour contents into a 5ml tube with cap
4) With a pipette, add 1ml of lysis solution (detergent) - lyses the cells by disruption the bonds of the lipids and protein that compose the membranes
5) Invert the tube 5 times (mixes the cell lysis solution with the cells) - gently or else the long strands of DNA will break - sit for 2 mins
6) Hold tube at 45 angle, add 70% ethanol to fill the tube. DO NOT MIX!
7) Translucent DNA will clump in where the layers meet - DNA precipitates
8) Sit for 10 mins - DNA will continue to precipitate out of solution and extend through the ethanol

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12
Q

In the first step of Extraction, why don’t we use water as a medium of collection of DNA?

A

The water would be hypotonic to the cells, therefore the cells would burst and release their DNA prior to collection

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13
Q

When we add the 70% ethanol which two layers do we see?

A

Upper - ethanol layer

Lower - cheek cell lysate

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14
Q

What allows the DNA to precipitate?

A

The Gatorade (NaCl), Sodium ions are + charged when in solution and are attracted to the - charged DNA. This attraction neutralizes the charge of DNA, allowing it to stick together during precipitation

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15
Q

Define Step 2: Polymerase Chain Reaction (PCR)

A

Process that rapidly makes identical copies of the DNA collected, which are then used in subsequent analysis

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16
Q

What are the 4 ingredients required for PCR?

A

1) DNA extract
2) 4 Deoxyribonucleotide triphosphates (dNTPs)
3) Primers
4) DNA Polymerase

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17
Q

Define Primers

A

Short segments of synthetic DNA necessary for the initiation of DNA replication

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18
Q

Define DNA Polymerase

A

A heat stable enzyme that elongated the DNA chain by adding dNTPs to the primers

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19
Q

What are the 3 steps in each PCR cycle?

Explain them

A

1) DNA denaturation (strand seperation) - Heat to separate the two strands of DNA (double helix)
2) Anneal primers (primer binding) - Cool so primers can bond to the single DNA strands
3) Extend primers (DNA synthesis) - Heat so DNA (Taq) polymerase can add dNTPs to the end of the primers

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20
Q

Define Step 3: Restriction Fragment Analysis

A

Enables an indirect comparison of nucleotide sequences in different amplified DNA samples through the use of restriction enzymes with cut the DNA within specific recognition sequences/ restriction sites

21
Q

Which restriction enzyme do we use in this experiment? What does it do?
What are the resulting lengths called?

A

EcoR1
It recognizes and cuts DNA within the nitrogenous base sequence GAATTC
- Restriction fragments

22
Q

What are the two steps within Step 3: Restriction Fragment Analysis?

A

i) Restriction Digest

ii) Gel electrophoresis

23
Q

Step 3 i) Restriction Digest

A

The restriction enzyme (EcoR) is added to the PCR product and the solution is place in an incubator
- Cuts DNa into specific fragment sizes and numbers

24
Q

Step 3 ii) Gel electrophoresis

A

A process that allows us to separate the restriction fragments produced by each digested PCR into an ordered group (based on size & differences)

25
Q

What is the Gel electrophoresis based on?

A

Based on the - charge of DNA & the differences in size of the DNA fragments
- smaller fragments travel further in the gel than larger fragments

26
Q

In the experiment which type of gel did we use?

A

Gels made of agarose (a polysaccharide derived from algae)

27
Q

How do we complete a gel electrophoresis experiment?

A

1) Put the gel into electrophoresis chamber (containing salt water which conducts electricity)
2) Samples containing restriction fragments are loaded into well in the gel
3) Attach electrodes to the chamber and current is applied
4) The (-) DNA migrates towards the (+) electrode
5) Once the samples have migrated sufficiently the gel is stained with a gel that binds to the DNA so that the restriction fragments can be visualized

28
Q

Step 4: Interpreting the results

What is the ladder?

A

Contains DNA fragments of known size, which are used to estimate the size of the unknown DNA fragments at the crime scene

29
Q

Recombinant DNA

A

DNA from two different sources is combined into one molecule

  • Uses restriction enzymes
  • Could use it to produce crops that are resistant to herbicides, pests, disease
30
Q

Genetically modified organism (GMO)

A

An organism that has acquired genes through any artificial process

31
Q

Transgenic organism

A

If the genes are from another species

- Roundup Ready Canola which is resistant to the herbicide Roundup

32
Q

Roundup Ready Canola is a ______ organism because it has genes from another species

A

Transgenic

33
Q

Restriction enzymes are used in which 2 techniques?

A

Paternity testing & Recombinant DNA

34
Q

What charge does DNA carry? Why?

A

Negative charge because of phosphate group

35
Q

List the 3 solution used in DNA extraction and briefly describe their functions

A

1) Gatorade (Saline solution) = Isotonic solution so the cells will not burst & release DNA prior to collection
2) Cell lysis solution (Green) = breaks open the plasma and nuclear membranes to release the DNA
3) Ethanol (70%)= DNA is insoluble in alcohol so it precipitates out of the solution and into the ethanol layer

36
Q

After DNA extraction, what are the 3 procedures that are used before you can interpret evidence from a crime scene?

A

1) Polymerase Chain Reaction (PCR)
2) Restriction Digest
3) Gel Electrophoresis

37
Q

What does PCR stand for?

A

Polymerase Chain Reaction

38
Q

List the 4 ingredients used in PCR

A

1) DNA Extract
2) Four dNTPs
3) Primers
4) DNA Polymerase (enzyme)

39
Q

What type of enzyme is used to cut the DNA into fragments during restriction digest?

A

Restriction enzymes

We used EcoR1 & BamH1

40
Q

What is the specific sequence of DNA that EcoR1 recognizes and binds to in order to cut the DNA?

A

GAATTC

41
Q

The principle of gel electrophoresis is based on the negative charge of the ____ and the differences in _____ of the DNA fragments, with the ______ fragments traveling farther in the gel than the ______ fragments. The gel is submerged in a chamber containing salt water because it __________. The DNA fragments move towards the _____ end of the gel.

A

The principle of gel electrophoresis is based on the negative charge of the DNA and the differences in size of the DNA fragments, with the smaller fragments traveling farther in the gel than the larger fragments. The gel is submerged in a chamber containing salt water because it conducts electricity. The DNA fragments move towards the positive end of the gel.

42
Q

What makes the DNA fragments visible in the gel?

A

The gel is stained with a special stain that binds to the DNA

43
Q

T or F:
In crime scene investigations, restriction fragments of several genes (10 or 13) are analyzed to decrease the possibility of two individuals having identical patterns of restriction fragments

A

True

44
Q

T or F:

Genes have many different alleles and the prevalence of each allele varies among different ethnic groups

A

True

45
Q

T of F:

The pattern of restriction fragments in a gel is called a DNA footprint

A

False; fingerprint

46
Q

T or F:

In paternity testing, DNA is collected from cells that lack a nucleus

A

False; animal cells have a nucleus

47
Q

T or F:

Recombinant DNA is one DNA molecule that has been engineered from two different sources

A

True

48
Q

Why are some of the bands of DNA fragments thicker (brighter) than the others?

A

Because of the alleles of a gene vary in their nucleotide sequences

49
Q

List 3 examples of modern biotechnology & how they benefit humans

A

1) Medicine - Recombinant bacteria are used to produce biosynthetic insulin & other disease specific medicines
2) Transgenic plants - Genetic modification of plants
3) Bioremediation - Microorganisms are used to clean up the pollutants in the environment

Bio 120 Lab (4 decks)

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