(SBI4U1) Molecular Biology Techniques Flashcards
Why is determining the sequence of DNA important? (4)
Solve crimes using DNA analysis
Determine paternity suits
Fetal screening for genetic disorders
Research to cure/treat diseases
How do we sequence DNA? (3)
Break DNA in small pieces at specific sites
Copy or amplify DNA to et a large sample
Sort and analyse DNA
How do we break DNA?
Can be cut into small fragments using restriction enzymes
What do restriction enzymes do?
Recognize specific sequences of DNA and cut the DNA at these sites
What are the sites the restriction enzymes cut the DNA at called?
Restricition sites
What are restriction enzymes used for?
Used by prokaryotes to defend themselves against infection
Restriction sites can now be used to _______ specific sequences of _______ that need to be _______
Isolate
Interest
Studied
How do we amplify DNA?
We can make many copies of DNA segments by cloning using bacterial DNA call vectors
What are bacterial vectors?
Circular portions of DNA
What are bacterial vectors also known as?
Plasmids
How is amplifying DNA by cloning using bacterial vectors accomplished? (3)
By cutting open the bacterial DNA (plasmid)
Cutting out the DNA sequence of interest using the same restriction enzyme
Inserting the DNA sequence cut out into the plasmid
Why can DNA sequence be inserted into the cut plasmid?
Restriction enzymes leave sticky ends that are complementary on the bacterial plasmid and DNA of interest, thus when joined they base pair and stick together
What is the Target DNA + Plasmid known as?
Recombinant DNA
What is done to the recombinant plasmid?
Put back into the bacteria, to be multiplied as the bacteria reproduce
What does PCR stand for?
Polymerase Chain Reaction
What are the ingredients in PCR? (4)
DNA
Primers
Nucleotides
DNA polymerase
What are the steps of PCR? (3)
Target DNA is heated (90 degrees Celsius) to separate DNA strands
Cooled (37 degrees Celsius) and primers are added to each DNA strand (starting point for DNA polymerase)
Heated to 70 degrees Celsius to activate DNA polymerase to attach bases to primer sequence, making 2 identical DNA strands
Cycle repeats
What are the applications of amplifying DNA? (6)
Fetal screening Analysis of mutations Diagnosis of genetic disorders (Huntington's, CF, DMD) Detection of microorganisms Parental disputes Forensic Science (DNA evidence)
What is the main method of sorting and analysing DNA?
Gel Electrophoresis
What does gel electrophoresis do?
Separates molecules according to size and electrical charge
Gel electrophoresis:
___ fragments travel through a special gel towards a positive charge (DNA has a net ________ charge)
Gel is ______, so _____ DNA fragments can travel farther than _____ fragments
DNA Negative Porous Small Large
Gel electrophoresis:
___ fragments separated by gel electrophoresis will generate a specific pattern of _____ called a ___ ___________
DNA
Bands
DNA fingerprint
True or false:
Each person’s DNA fingerprint is the same
False, each person’s DNA fingerprint is unique
What are the steps in gel electrophoresis? (4)
Restriction enzymes cleave DNA into smaller segments of various sizes
DNA segments are loaded into wells in a porous gel. The gel floats in a buffer solution within a chamber between two electrodes
When an electric current is passed through the chamber, DNA fragments move toward the positively-charged cathode
Smaller DNA segments move faster and farther than larger DNA segments
What can DNA fingerprinting be used for? (2)
Parental disputes
Matching DNA at crime scenes to suspects
What can gel electrophoresis be used for?
Research purposes (identifying target genes in disease, testing drugs, identifying conditions for disease progression)
