Lab 7 Flashcards
Biotechnology
The use of organisms or their components (DNA, protein) to make or modify products useful to humans
Traditional Biotechnology
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
Modern Biotechnology
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
What has Biotechnology enabled?
Advances in forensics, medicine, agriculture, pollution control, evolutionary biology
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 __________)
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)
Central dogma
The flow of information from DNA to RNA to protein
- RNA makes polypeptides into amino acids
Biological samples collected for DNA analysis must contain _____ cells
EX?
Nucleated cells
EX. bodily fluids, hair, skin, organs, bones
What typed of biological samples are most commonly obtained from suspects for analysis?
Blood, hair, buccal (cheek) cells
What are the steps in processing and analyzing DNA collected from a crime scene?
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
Step 1: DNA Extraction
Define Extraction
- 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
What are the 8 steps of Extraction from our cheek cells? And why do we do them?
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
In the first step of Extraction, why don’t we use water as a medium of collection of DNA?
The water would be hypotonic to the cells, therefore the cells would burst and release their DNA prior to collection
When we add the 70% ethanol which two layers do we see?
Upper - ethanol layer
Lower - cheek cell lysate
What allows the DNA to precipitate?
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
Define Step 2: Polymerase Chain Reaction (PCR)
Process that rapidly makes identical copies of the DNA collected, which are then used in subsequent analysis
What are the 4 ingredients required for PCR?
1) DNA extract
2) 4 Deoxyribonucleotide triphosphates (dNTPs)
3) Primers
4) DNA Polymerase
Define Primers
Short segments of synthetic DNA necessary for the initiation of DNA replication
Define DNA Polymerase
A heat stable enzyme that elongated the DNA chain by adding dNTPs to the primers
What are the 3 steps in each PCR cycle?
Explain them
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
Define Step 3: Restriction Fragment Analysis
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
Which restriction enzyme do we use in this experiment? What does it do?
What are the resulting lengths called?
EcoR1
It recognizes and cuts DNA within the nitrogenous base sequence GAATTC
- Restriction fragments
What are the two steps within Step 3: Restriction Fragment Analysis?
i) Restriction Digest
ii) Gel electrophoresis
Step 3 i) Restriction Digest
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
Step 3 ii) Gel electrophoresis
A process that allows us to separate the restriction fragments produced by each digested PCR into an ordered group (based on size & differences)
What is the Gel electrophoresis based on?
Based on the - charge of DNA & the differences in size of the DNA fragments
- smaller fragments travel further in the gel than larger fragments
In the experiment which type of gel did we use?
Gels made of agarose (a polysaccharide derived from algae)
How do we complete a gel electrophoresis experiment?
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
Step 4: Interpreting the results
What is the ladder?
Contains DNA fragments of known size, which are used to estimate the size of the unknown DNA fragments at the crime scene
Recombinant DNA
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
Genetically modified organism (GMO)
An organism that has acquired genes through any artificial process
Transgenic organism
If the genes are from another species
- Roundup Ready Canola which is resistant to the herbicide Roundup
Roundup Ready Canola is a ______ organism because it has genes from another species
Transgenic
Restriction enzymes are used in which 2 techniques?
Paternity testing & Recombinant DNA
What charge does DNA carry? Why?
Negative charge because of phosphate group
List the 3 solution used in DNA extraction and briefly describe their functions
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
After DNA extraction, what are the 3 procedures that are used before you can interpret evidence from a crime scene?
1) Polymerase Chain Reaction (PCR)
2) Restriction Digest
3) Gel Electrophoresis
What does PCR stand for?
Polymerase Chain Reaction
List the 4 ingredients used in PCR
1) DNA Extract
2) Four dNTPs
3) Primers
4) DNA Polymerase (enzyme)
What type of enzyme is used to cut the DNA into fragments during restriction digest?
Restriction enzymes
We used EcoR1 & BamH1
What is the specific sequence of DNA that EcoR1 recognizes and binds to in order to cut the DNA?
GAATTC
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.
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.
What makes the DNA fragments visible in the gel?
The gel is stained with a special stain that binds to the DNA
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
True
T or F:
Genes have many different alleles and the prevalence of each allele varies among different ethnic groups
True
T of F:
The pattern of restriction fragments in a gel is called a DNA footprint
False; fingerprint
T or F:
In paternity testing, DNA is collected from cells that lack a nucleus
False; animal cells have a nucleus
T or F:
Recombinant DNA is one DNA molecule that has been engineered from two different sources
True
Why are some of the bands of DNA fragments thicker (brighter) than the others?
Because of the alleles of a gene vary in their nucleotide sequences
List 3 examples of modern biotechnology & how they benefit humans
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
