Lecture 24 Flashcards
Define Genetic variation:
How can it be identified? (3)
Differences in DNA sequences (genome) of individuals of a species brought about by mutation.
May be identified by
1. Phenotype
- Protein differences
- DNA sequences differences (polymorphisms)
How can Genetic Variation be identified for
- DNA sequence differences polymorphism
DNA sequence differences polymorphism:
—> Genetic markers (aka molecular markers)
———————-> May not code for any physical trait, but can be detected through molecular techniques
** Remember variations in genes may not alter a protein’s function.
SNP =
Single nucleotide polymorphism = SNP
Note: to be classified as a genetic polymorphism, the rarest type cannot be maintained by recurrent random mutation, i.e. it has to have an appreciable frequency (typically 1%).
Analysis of Genetic Markers: WHAT ARE THE THREE GENERAL CATEGORIES OF TECHNIQUES
- Restriction enzyme-based, for example:
** restriction fragment length polymorphism (RFLP). - Polymerase chain reaction (PCR)-based, for example:
** Random amplification of polymorphic DNA (RAPD)
** microsatellite - DNA sequencing, for example:
** Single nucleotide polymorphism (SNP)
GENETIC MARKERS - RFLPs: Restriction Enzymes; dot point of steps
- Restriction Enzymes:
- Agarose Gel Electrophoresis:
- Southern Blotting:
GENETIC MARKERS - RFLPs: Restriction Enzymes; steps in detail
Restriction Enzymes:
- Digest double-stranded DNA at internal phosphodiester bonds.
- Cut DNA at specific recognition sites called restriction enzyme palindromes.
- Palindromes are sequences of 4-8 nucleotides that read the same on both DNA strands.
- Cuts result in fragments with 3’-hydroxyl and 5’-phosphate groups.
- Types of cuts:
- Blunt ends: Cuts that leave no overhangs.
- Sticky ends (cohesive ends): Cuts that leave overhangs, either 5’- or 3’-overhangs.
Agarose Gel Electrophoresis:
- Gel electrophoresis technique using agarose, a red algal carbohydrate.
- Agarose is heated in a buffer to dissolve and poured into a gel mold.
- DNA carries a negative charge and migrates towards the positive electrode.
- Molecules are separated based on size.
- DNA fragments are visualized by staining with a fluorescent dye or radioisotope.
- Molecular size standards (DNA ladder) are used to determine fragment sizes.
Southern Blotting:
- Technique to transfer DNA fragments from a gel to nitrocellulose paper.
- Nitrocellulose paper is single-stranded, allowing DNA to bind to it.
- Process:
1. DNA fragments are separated by agarose gel electrophoresis.
2. Gel is placed on the nitrocellulose paper and covered with a buffer.
3. A labeled (e.g., radioactive) single-stranded DNA probe is added to the buffer.
4. The probe hybridizes (binds) to complementary DNA sequences on the nitrocellulose.
5. Autoradiography or other methods visualize DNA fragments that bind to the probe.
Explain GENETIC MARKERS - RFLPs: Restriction Enzymes;step 1
- Digest double-stranded DNA at internal phosphodiester bonds.
- Cut DNA at specific recognition sites called restriction enzyme palindromes.
- Palindromes are sequences of 4-8 nucleotides that read the same on both DNA strands.
- Cuts result in fragments with 3’-hydroxyl and 5’-phosphate groups.
- Types of cuts:
- Blunt ends: Cuts that leave no overhangs.
- Sticky ends (cohesive ends): Cuts that leave overhangs, either 5’- or 3’-overhangs.
Explain GENETIC MARKERS - RFLPs: Restriction Enzymes; Step 2.
Agarose Gel Electrophoresis:
- Gel electrophoresis technique using agarose, a red algal carbohydrate.
- Agarose is heated in a buffer to dissolve and poured into a gel mould.
- DNA carries a negative charge and migrates towards the positive electrode.
- Molecules are separated based on size.
- DNA fragments are visualized by staining with a fluorescent dye or radioisotope.
- Molecular size standards (DNA ladder) are used to determine fragment sizes.
Explain GENETIC MARKERS - RFLPs: Restriction Enzymes; Step 3.
Southern Blotting:
- Technique to transfer DNA fragments from a gel to nitrocellulose paper.
- Nitrocellulose paper is single-stranded, allowing DNA to bind to it.
- Process:
1. DNA fragments are separated by agarose gel electrophoresis.
2. Gel is placed on the nitrocellulose paper and covered with a buffer.
3. A labeled (e.g., radioactive) single-stranded DNA probe is added to the buffer.
4. The probe hybridizes (binds) to complementary DNA sequences on the nitrocellulose.
5. Autoradiography or other methods visualize DNA fragments that bind to the probe.
What is RFLP Analysis? (5)
- SEPARATION of fragments by agarose gel electrophoresis
- TRANSFER (blotting) of the fragments to nitrocellulose
- LABELLING the DNA on the nitrocellulose with a known probe - in this example, the fragment from the ancestral chromosome
- DETECTION of labelled DNA fragments e.g. autoradiography
- Can DETECT SNPs
GENETIC MARKERS - RAPDs, Microsatellites: Dependent on the PCR
For the PCR need: 2 things, why, how?
For the PCR need:
- Primers
- need sequence information to design
- 20-30 nucleotides in length
- bind complementary sequences on the template DNA strands, e.g. flank region to be amplified. - DNA Polymerase
- heat stable, e.g. from thermophilic bacteria, THERMUS AQUATICUS (Taq) polymerase.
What is Taq polymerase?
Taq polymerase is from Thermus aquaticus, isolated from the hot springs in Yellowstone National Park by Thomas Brock. PCR was invented by Kary Mullis.
Genetic Markers - RAPD, micro-satellite
For the PCR, also need:
- DNA to be copied (aka template)
- dATP, dGTP, dTTP (dNTPs)
- buffer
- Thermocycler
- heating, cooling
- the timing of cycles
eg. after 20-30 cycles —> 2^20 - 2^30 copies of original template
Genetic Markers - RAPDs – Explain it, cause of polymorphsism
For RAPDs - primers are random sequences of about 10 bases
- use multiple primer pairs on the same template DNA, BUT ONLY 1 PRIMER PAIR per PCR.
Individual 1.
——————> combine PCRs, and agarose gel electrophoresis = standards
individual 2.
Polymorphism due to:
- no primer binding site (X)
- Variable distance between primer binding sites
Genetic markers - microsatellites? what is it?
Microsatellites - variable number of tandem repeats (VNTRs) of a small number of nucleotides, e.g. a dinucleotide such as CA
Note: you will use similar PCR -based techniques in pra 3 using primers specific to the human PV92 locus.
