lecture 4- molecular techniques

Question 1

name a few general types of mutagens

Answer 1

1- chemical- EMS

2- high energy- xray, gamma ray

3- insertional- transposons (transposable elements), T-DNA (transfer-DNA), homologous recombination- CRISPR*

Question 2

how does EMS work as a mutagen?

Answer 2

usually results in point mutations: single bp transitions (G/C - A/T)

Question 3

how do x-rays and other high energy sources work as mutagens?

Answer 3

usually results in major DNA rearrangements such as translocations, large duplications, inversions, and deficiencies

Question 4

how do transposons and insertional mutagens work as mutagens? (2 effects)

Answer 4

1- large segments of DNA often many thousands of bases (Kb) that disrupt gene function - major effect

2- small deletions (a few bp) caused by transposon excisions or hopping (minor- potential effect)

Question 5

mutagenesis and mutant screens is used to study the ___

Answer 5

gene or phenotype of your interest

Question 6

mutagenesis and mutant screens requires generating a ___ and then screening through ___

Answer 6

mutant population
through this population for mutants that the gene responsible can then be determined

Question 7

how mutagenesis works- you are targeting the entire ___ of an organism and you need to control several things

Answer 7

genome

Question 8

what things do you need to control in mutagenesis?

Answer 8

mutation rate:
- too large = lethal
- too small = hard to find
- about one mutant per organism = just right

screening mechanism (finding what you want made easy)

Question 9

mutant screens- forward vs. reverse genetics (gene & phenotype)-

Answer 9

forward genetics screen = you know the phenotype you want, going after the gene responsible

reverse genetics screen = you know the gene you want, going after the phenotype involved (mutating the gene to find what function it has)

Question 10

screening issues- phenotype edition

Answer 10

• how many can you screen (numbers)
• how distinct (is your phenotype 100% related to what you are after)
• how easy (effort required to conduct)
• did someone else do this already

Question 11

screening issues- gene edition

Answer 11

essential (required) genes vs. non-essential genes

redundant genes (multiple copies) vs. single copy genes

physical gene size- large vs. small

Question 12

name the positives and negatives of forward genetics screens

Answer 12

you are screening mutants for a specific phenotype to better understand it, by finding/determining the genes involved

+ no prior knowledge of sequence or even the gene to be mutated is needed
+ should always find your gene to study

• redundancy is problematic
• hard to know what you will get

Question 13

name the positives and negatives of reverse genetic screens

Answer 13

you are looking to mutate a specific gene (a targeted mutagenesis approach) to understand function of altered phenotype

+ need to have some sequence knowledge of your gene
- phenotypes that are produced are unknown (can be lethal or not obvious)

Question 14

name 5 ways transgenics are made

Answer 14

1- insertional mutagenesis
2- transposons
3- transfer-DNA
4- homologous recombination (site specific)
5- CRISPR (site specific)

Question 15

how are transgenics made through insertional mutagenesis?

Answer 15

get DNA integrated into the genome
- adding to embryo (microinjection)
- electroporation of cells (shocking)
- particle bombardment (shotgun)

Question 16

how are transgenics made through transposons?

Answer 16

• internal DNA that can jump or hop from one place in a genome to another
• used in all eukaryotes (P-elements in Drosophilia)
• “random”

Question 17

how are transgenics made through t-DNA?

Answer 17

• bacterial integration of DNA (plants only- mostly eudicots)
• “random”

Question 18

what gets put in a transgenic line?

Answer 18

1- a selectable marker so you can detect when you have generated a transgenic (most processes tend to be low yielding- single digit %’s)
- often antibiotic resistance, visible markers (GFP), other resistances (herbicides)

2- your fav gene (YFG)
- overexpressed- constitutively, inducibly
- promoter only (with trackable marker- LacZ, GFP, GUS)
- antisense or RNAi (gene or part of gene backwards to bind up all copies normally expressed)

Question 19

example of transfer-DNA transfer in plants

Answer 19

• the soil bacterial Agrobacteria tumefaciens, integrates a piece of its DNA into the plant genome- natural transgenic marker
• scientists removed the bacteria gene and put in genes that they wanted YFG and antibiotic resistance genes