Lecture 29 - Molecular biology techniques

Question 1

Function of interspersed repeats for genome evolution (2)

Answer 1

Gene duplication

Exon shuffling

Question 2

3 mechanisms of evolutionary gene duplication (3 scales)

Answer 2

1) Whole genome duplication (tetraploidization)
2) Duplication of entire chromosomes
3) Duplication of segments within chromosomes (partial)

Question 3

How can intersperesed repeats drive gene duplication and duplication of segments within a chromosome

Answer 3

Unequal crossing over during homologous recombination for a gene surrounded by 2 identical interspersed repeats

Question 4

Most common LINE (long interspersed element and 2 other

Answer 4

LI, (L2,L3)

Question 5

What is a gene family and what differentiates genes in a gene family

Answer 5

Related genes that descend from ancestral gene but that diverged in sequence and function

Question 6

Def. of exon shuffling

Answer 6

Idea that exons can move from gene to gene over evolutionary time

Question 7

Why is it possible to shuffle exons to make new exon combinations for new proteins

Answer 7

Because exons can code for protein domains (which work indep.) so can be shuffled

Question 8

What can drive exon shuffling

Answer 8

Double crossover between 2 genes because of identical interspersed repeats found at 2 places in the 2 genes between exons

Question 9

What is Alu

Answer 9

Most common SINE and most common mobile element in human genome (1 million)

Question 10

Name of the technique that can help predict a protein’s function and program used

Answer 10

Bioinformatics. BLAST (Basic local alignement search tool) program

Question 11

What BLAST does to predict a protein’s function

Answer 11

Compares its sequence to all known protein sequences (it derives them from the genome) and finds protein with most similarity

Question 12

Example of protein + its function that were found by bioinformatics + which protein it was based on

Answer 12

NF1 (unknown function) had similarity with Ira (a GAP for Ras). NF1 turned out to have the same function

Question 13

Additional genomes of eukaryotes

Answer 13

Genome in mitochondria and chloroplasts

Question 14

What do mitochondria and chloroplasts originate from

Answer 14

Endosymbiotic bacteria

Question 15

How much of ancestral bacterial genome (of mito/chloro) is retained

Answer 15

Part of the ancestral bacterial genome is retained (so it’s part of our genome/we still have some of it)

Question 16

T/F : Diseases only arise from DNA in the nucleus

Answer 16

False : Diseases may arise from mitochondrial DNA

Question 17

DNA fragments GEL electrophoresis : On what base fragments are separated + 2 possible gels

Answer 17

Separation by SIZE. Agarose or acrylamide gel

Question 18

Dye used for visualization of DNA electrophoresis gel

Answer 18

ethidium bromide (fluorescent DNA-binding dye)

Question 19

Electrophoresis of a mixture vs GEL electrophoresis with of a mixture

Answer 19

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

Question 20

Amplification of specific DNA fragments : Name of technique

Answer 20

PCR : Polymerase chain reaction

Question 21

PCR in vitro or in vivo + principle

Answer 21

in vitro. multiple replication cycles and double amount of DNA in each cycle

Question 22

Advantage of PCR amplification

Answer 22

We can isolate a single gene in pure form

Question 23

What happens if we start PCR with 1 kb molecule and do 35 cycles

Answer 23

Obtain 40 ng of DNA

Question 24

What is required in solution for a PCR

Answer 24

primers, templates and NTPs (nucleotide triphosphates)

Question 25

Primer description + how is designed

Answer 25

short synthetic oligonucleotide (15-50 bp) designed to fit gene of interest

Question 26

Differences of PCR vs in vivo DNA replication (3)

Answer 26

No helicase, no replication fork, no lagging strand

Question 27

3 steps of PCR

Answer 27

Denaturation of DNA, annealing of primers, elongation

Question 28

Denaturation step temperature

Answer 28

95 C high temp to separate strands

Question 29

Primer annealing step temperature/why

Answer 29

50-60 C. Done at temp. high enough to permit only perfect primer matching

Question 30

Elongation/DNA synthesis step temperature/why

Answer 30

72 C, done at the optimal temp. for thermostable DNAP

Question 31

Necessary characteristic for DNAP and where it will be taken from

Answer 31

Need to be thermostable. Will use DNAP from prokaryotes living in hot springs

Question 32

Example of DNAP and species it comes from

Answer 32

Taq DNA polymerase (thermus aquaticus)

Question 33

What is RT-PCR + its goal

Answer 33

Reverse transcriptase PCR. Goal is to detect mRNAs

Question 34

Where do we get a reverse transcriptase from

Answer 34

From a retrovirus

Question 35

Steps for RT-PCR (3)

Answer 35

Extract RNA from cells/tissues (mixture of all RNAs), reverse transcriptase to obtain mixture of cDNA, PCR using specific primers

Question 36

What RT-PCR can allow us to study (2 examples)

Answer 36

Alternative splicing, variation in mRNA within diff. cells

Question 37

2 possible modifications to primers

Answer 37

1) Add nts to 5’ end

2) Introduce point mutation

Question 38

Utility of adding nts to 5’ end of primer

Answer 38

Added nucleotides won’t hybridize w/ template but will be conserved and serve to create a RESTRICTION ENZYME SITE, which is useful

Question 39

Utility of introducing a point mutation in a primer

Answer 39

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

Question 40

Old DNA sequencing method name + what it does

Answer 40

Sanger sequencing -> sequences one DNA per reaction

Question 41

Advantage of Sanger sequencing

Answer 41

Low cost so useful for sequencing small number of DNAs

Question 42

Next generation sequencing method name

Answer 42

Illumina platform

Question 43

Two steps of illumina platform

Answer 43

1) Generate millions of microscopic DNA spots on glass slide

2) Sequencing steps

Question 44

Generate microscopic DNA spots : Step 1 (what is used and DNA charact.)

Answer 44

Use dilute DNA and glass with covalently attached primers. dsDNA has ligate linkers on one end

Question 45

Generate microscopic DNA spots : Step 2

Answer 45

Denature DNA strand, hybridize it to primer and elongate primer w/ Taq DNAP

Question 46

Generate microscopic DNA spots : Step 3 (after a first primer is elongated)

Answer 46

Denature dsDNA (extended primer remains), anneal to other primer with ligate linker and extend other primer

Question 47

Generate microscopic DNA spots : Step 4 (how many times cycle done and name of end result)

Answer 47

10 cycles lead to a polony (PCR colony) or approx 1000 identical strands (each spot is a polony)

Question 48

Sequencing steps : Step 1

Answer 48

Cut 2nd primer covalently linked to glass and add 2nd primer (now binds to standing DNA)

Question 49

Sequencing steps : Step 2

Answer 49

Add fluorescently labeled dNTPs and check colours on of glass spots on fluorescent imaging

Question 50

What is particular about the fluorescently labeled dNTPs

Answer 50

fluorophore is added to 3’-OH so DNAP can’t add to that

Question 51

Sequencing steps : Step 3

Answer 51

Remove chemically the fluorescent tag on the newly added dNTP and add another fluorescent dNTP (restart)

Question 52

How many times sequencing steps usually repeated

Answer 52

approx. 100 times