Lecture 29 - Molecular biology techniques Flashcards
Function of interspersed repeats for genome evolution (2)
Gene duplication
Exon shuffling
3 mechanisms of evolutionary gene duplication (3 scales)
1) Whole genome duplication (tetraploidization)
2) Duplication of entire chromosomes
3) Duplication of segments within chromosomes (partial)
How can intersperesed repeats drive gene duplication and duplication of segments within a chromosome
Unequal crossing over during homologous recombination for a gene surrounded by 2 identical interspersed repeats
Most common LINE (long interspersed element and 2 other
LI, (L2,L3)
What is a gene family and what differentiates genes in a gene family
Related genes that descend from ancestral gene but that diverged in sequence and function
Def. of exon shuffling
Idea that exons can move from gene to gene over evolutionary time
Why is it possible to shuffle exons to make new exon combinations for new proteins
Because exons can code for protein domains (which work indep.) so can be shuffled
What can drive exon shuffling
Double crossover between 2 genes because of identical interspersed repeats found at 2 places in the 2 genes between exons
What is Alu
Most common SINE and most common mobile element in human genome (1 million)
Name of the technique that can help predict a protein’s function and program used
Bioinformatics. BLAST (Basic local alignement search tool) program
What BLAST does to predict a protein’s function
Compares its sequence to all known protein sequences (it derives them from the genome) and finds protein with most similarity
Example of protein + its function that were found by bioinformatics + which protein it was based on
NF1 (unknown function) had similarity with Ira (a GAP for Ras). NF1 turned out to have the same function
Additional genomes of eukaryotes
Genome in mitochondria and chloroplasts
What do mitochondria and chloroplasts originate from
Endosymbiotic bacteria
How much of ancestral bacterial genome (of mito/chloro) is retained
Part of the ancestral bacterial genome is retained (so it’s part of our genome/we still have some of it)
T/F : Diseases only arise from DNA in the nucleus
False : Diseases may arise from mitochondrial DNA
DNA fragments GEL electrophoresis : On what base fragments are separated + 2 possible gels
Separation by SIZE. Agarose or acrylamide gel
Dye used for visualization of DNA electrophoresis gel
ethidium bromide (fluorescent DNA-binding dye)
Electrophoresis of a mixture vs GEL electrophoresis with of a mixture
Electrophoresis of mixture alone = separation based on charge/mass ratio
GEL electrophoresis of mixture = we do it cause everything in mixture has same z/m ratio so separation done by SIZE
Amplification of specific DNA fragments : Name of technique
PCR : Polymerase chain reaction
PCR in vitro or in vivo + principle
in vitro. multiple replication cycles and double amount of DNA in each cycle
Advantage of PCR amplification
We can isolate a single gene in pure form
What happens if we start PCR with 1 kb molecule and do 35 cycles
Obtain 40 ng of DNA
What is required in solution for a PCR
primers, templates and NTPs (nucleotide triphosphates)
Primer description + how is designed
short synthetic oligonucleotide (15-50 bp) designed to fit gene of interest
Differences of PCR vs in vivo DNA replication (3)
No helicase, no replication fork, no lagging strand
3 steps of PCR
Denaturation of DNA, annealing of primers, elongation
Denaturation step temperature
95 C high temp to separate strands
Primer annealing step temperature/why
50-60 C. Done at temp. high enough to permit only perfect primer matching
Elongation/DNA synthesis step temperature/why
72 C, done at the optimal temp. for thermostable DNAP
Necessary characteristic for DNAP and where it will be taken from
Need to be thermostable. Will use DNAP from prokaryotes living in hot springs
Example of DNAP and species it comes from
Taq DNA polymerase (thermus aquaticus)
What is RT-PCR + its goal
Reverse transcriptase PCR. Goal is to detect mRNAs
Where do we get a reverse transcriptase from
From a retrovirus
Steps for RT-PCR (3)
Extract RNA from cells/tissues (mixture of all RNAs), reverse transcriptase to obtain mixture of cDNA, PCR using specific primers
What RT-PCR can allow us to study (2 examples)
Alternative splicing, variation in mRNA within diff. cells
2 possible modifications to primers
1) Add nts to 5’ end
2) Introduce point mutation
Utility of adding nts to 5’ end of primer
Added nucleotides won’t hybridize w/ template but will be conserved and serve to create a RESTRICTION ENZYME SITE, which is useful
Utility of introducing a point mutation in a primer
Primer will still anneal and remain in place. Half of new strands will lead to proteins with a different amino acid and other half will lead to wild-type protein
Old DNA sequencing method name + what it does
Sanger sequencing -> sequences one DNA per reaction
Advantage of Sanger sequencing
Low cost so useful for sequencing small number of DNAs
Next generation sequencing method name
Illumina platform
Two steps of illumina platform
1) Generate millions of microscopic DNA spots on glass slide
2) Sequencing steps
Generate microscopic DNA spots : Step 1 (what is used and DNA charact.)
Use dilute DNA and glass with covalently attached primers. dsDNA has ligate linkers on one end
Generate microscopic DNA spots : Step 2
Denature DNA strand, hybridize it to primer and elongate primer w/ Taq DNAP
Generate microscopic DNA spots : Step 3 (after a first primer is elongated)
Denature dsDNA (extended primer remains), anneal to other primer with ligate linker and extend other primer
Generate microscopic DNA spots : Step 4 (how many times cycle done and name of end result)
10 cycles lead to a polony (PCR colony) or approx 1000 identical strands (each spot is a polony)
Sequencing steps : Step 1
Cut 2nd primer covalently linked to glass and add 2nd primer (now binds to standing DNA)
Sequencing steps : Step 2
Add fluorescently labeled dNTPs and check colours on of glass spots on fluorescent imaging
What is particular about the fluorescently labeled dNTPs
fluorophore is added to 3’-OH so DNAP can’t add to that
Sequencing steps : Step 3
Remove chemically the fluorescent tag on the newly added dNTP and add another fluorescent dNTP (restart)
How many times sequencing steps usually repeated
approx. 100 times
