Manipulating DNA
Learning Objectives:
By the end of this article you should be able to:
- Explain how gel electrophoresis separates DNA fragments of different lengths
- List the main steps in PCR (polymerase chain reaction)
Gel Electrophoresis
Introduction to Gel Electrophoresis by Khan Academy
A technique used to separate DNA fragments (‘bits of DNA’) on the basis of their size/length. Gel electrophoresis can thus help us when we are comparing samples such as in forensic testing of DNA at crime scenes, and paternity testing (do we find DNA fragments of the same length?). Additionally, in laboratory research we can use it iscolate particular DNA fragments we are interested in (i.e. if we originally have a mix of fragments, but are only interested in one particular fragment).
Important Ideas:
- DNA is negatively charged (so attracted to positive end)
- Smaller DNA fragments move faster
- DNA fragments of the same length move at the same speed
Steps:
Can you correctly order the gel electrophoresis steps?
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Reveal gel electrophoresis steps
- DNA is extracted from cells/obtained from PCR
- Fragments of DNA are loaded into wells (in agar gel)
- The DNA is subjected to a electric current, with the negative end closest to the starting point of the DNA
- DNA fragments move toward the positively charged electrode (this process happens because DNA is negatively charged, because of the phosphate group in its backbone)
- Smaller fragments are more easily able to move through the pores in the agar gel and thus move quicker (travel a further distance)
- DNA is treated with a chemical which causes the molecule to fluoresce
- Size can be calculated (in kilobases) by comparing against a known industry standard
PCR (Polymerase Chain Reaction)
Steps in PCR Khan Academy Introduction
A technique for amplifying DNA fragments exponentially through many cycles of multiplication.
Amplification Questions
If you initially started with one DNA fragment, how many DNA fragements will you have …
After one round of PCR?
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After two rounds of PCR?
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Steps:
Reveal PCR steps
- D (enaturing)
- 90°
- hydrogen bonds between strands are broken which creates single stranded DNA molecules
- in other words, complementary base pairing is separated
- A (nnealing)
- 50°
- Primers are able to anneal/bind to the two DNA strands)
- E (longation)
- 72°
- Taq polymerase attaches to the primer
- It then moves along the two template strands of DNA synthesizing a new strand of DNA from each of the two template strands (using complementary base pairing) from free nucleotides
Required components of PCR
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Today’s TL;DR
- Gel electrophoresis is used to separate and compare DNA of different lengths
- Smaller fragments are more easily able to move through the pores in the agar gel and thus move quicker
- PCR is used to amplify DNA fragments
- Main steps: Denaturation, Anneal, and Elongation (which repeat in cycles)