Development Genetics

Question 1

development (2)

Answer 1

• zygote simple single cell -> adult with many cell types

- involves growth, differentiation, and regulation for all multicellular organisms

Question 2

development characteristics (3)

Answer 2

1. complex: cells have different identities at early stages of development
2. progressive: broad decisions are made more early, restricted ones made late
3. highly regulated: fates of cells are highly regulated through time and space

Question 3

forward genetics

Answer 3

• exploratory

Question 4

general steps in forward genetics (9)

Answer 4

1. choose a phenotype: specific, selectable, or easily recognizable
2. mutagenesis
3. saturation screen: find mutants with specific phenotype
4. carry out basic genetic analysis: dominant vs recessive; complementation analysis
5. crude map location of mutant genes: genetic crosses and PCR with molecular markers to find linkage with mutant gene
6. detailed phenotypic analysis of the mutant
7. epistasis analysis
8. fine map and identify mutant genes (positional cloning)
9. isolate cDNAs of the genes, sequence, and predict amino acid sequence of encoded protein
10. similarity search on blast database
11. determine how cloned gene regulates development: biochemical role, expression patterns, etc

Question 5

basic PCR steps (5)

Answer 5

1. add oligonucleotide primers
2. heat to separate strands
3. cool; primers anneal
4. heat to moderate T; DNA synthesis
5. repeat steps 2-4

Question 6

reverse genetics (3)

Answer 6

• manipulative
• manipulate certain genes to produce desired mutant phenotypes
• more common today

Question 7

molecular markers (3)

Answer 7

• site of heterozygosity: variation in DNA sequence
• heritable, but neutral and doesn’t produce visible trait
• detected by PCR
• used in linkage analysis

Question 8

positional cloning (2)

Answer 8

• recombination frequency determines the distance between AG and markers
• distance between x and y = (# recombinant chromosomes/total # chromosomes) x 100

Question 9

dNTP (2)

Answer 9

• natural structure

- can form phosphodiester bonds due to OH position on 2’ carbon

Question 10

ddNTP (3)

Answer 10

• artificial structure
• cannot form phosphodiester bonds due to loss of OH group at 2’ carbon
• will stop DNA synthesis randomly at sites meant for the ddNTP

Question 11

chain-termination sequencing method (3)

Answer 11

1. carry out sequencing reactions with each terminator base (ddATP, ddCTP, ddTTP, ddGTP)
2. separate product by gel electrophoresis
3. read out nucleotide sequence

Question 12

automated DNA sequencing (3)

Answer 12

1. carry out sequencing reaction in single tube by adding: primer, template, DNA polymerase, dNTPs, and differently fluorescently labelled ddNTPS
2. separate DNA fragments through electrophoresis
3. colour fluorescence of each fragment is detected and recorded

Question 13

complementary DNA (cDNA) (2)

Answer 13

• dsDNA synthesized from mRNA template

- reaction catalyzed by reverse transcriptase enzyme

Question 14

reverse transcriptase enzymes (4)

Answer 14

1. anneal to polyA tail of mRNA
2. reverse transcriptase copies mRNA into cDNA to form mRNA-cDNA complex
3. mRNA degraded
4. DNA polymerase copies cDNA to form dsDNA