BIOL 303 Lecture Notes - Lecture 1: Molecular Cloning, Gene Mapping, Protein Purification
14 May 2018
School
Department
Course
Professor
Lec 1
Class focus
- gene identification -> forward/reverse genetics in model species
- molecular cloning of a gene ->map-based cloning and confirmation of candidate gene
- study of gene expression: microarray, RT-PCR, northern, in situ, promotor-reporter gene fusion
- study of gene function: gene KO/knockdown to analyze gene function, RNA interference, over and
inappropriate expression of a gene
- protein localization and dynamics: GFP
- protein interactions: methods of detection
- protein purification: tag-based protein purification to quantify protein interaction in a complex
- protein characterization: SDS-PAGE and 2D-PAGE for post translational modification
- protein characterization: western blot
Gene identification – lec 1 info
- genes are normally identified in a genetic model org
^ easy to work with, grow fast, lots of progeny, small genome, most have sequence genomes, easy to be
genetically manipulated to make transgenic lines, have international community of scientists working on same
system thus can access more info
- Arabidopsis thaliana is a model species for plant bio research: small in size 20cm mature, can grow indoors in
a large number, short life cycle of 8-10 weeks, genome only have 140mbp, approx. 20,000 protein-coding
genes only, easy genetic manipulation
- saccharomyces cerevisiae (yeast) has been used as a model eukaryote: single-celled eukaryotic org thus rel
simple, undergoes mitosis every 90 mins, grows rapidly in simple medium, genome has 12.5mbp, only 6000
protein-coding genes, easy genetic manipulation
^ too simple to really be compared to humans or mammals, thus only used as a base
- forward genetics = classical genetics -> going from known phenotype to finding the gene resp. Used to id
genes acting in process of interest
^ look for individuals w defects in your process of interest, conduct a genetic analysis for mutated genes to
find the one/ones involved
- reverse genetics = modern approach -> going from gene to function. Requires prior knowledge of the
gee/its odig i ode to specifically isolate it only and thus find the corresponding phenotype/behavioural
changes
- can use natural mutations to study genetics but it would take too long thus mutagens used to increase fq of
finding mutations and thus number of genes identified
^ chemical mutagens (ex. ethylmethane sulfonate) -> point mutations - affects proteins through truncation or
loss of ke aa
^ radiation (ex. x-rays) -> leads to small or large deletions of DNA of even chrm breakage and rearrangement
^ insertional -> mutagenize genes by sticking a large chunk of DNA in the middle of it or its regulator
sequences ex. transposable elements, transferred-DNA
- to fid out fo e. hat gees otol the poess oue iteested i… ado utatig ad isolatio of
mutants w phenotype of interest in next gen and then genetic analysis
Lec 2 – Gene mapping/positional cloning
- use DNA we extracted from SHORTY mutant last week to do gene mapping
- context paper: identified a mutant, STOP1, in which root grows short in acidic conditions. Isolated mutant (1st
step), in order to id what gene in the genome was mutated. Then, perhaps clone the gene further to det its
role at a molecular level (2nd step)
- loate a gee that is utated i a gie geoe idetifig the distae btwn the mutated gene and
nearby landmarks, which could be either morphological or molecular markers
- morphological marker: gene which when mutated, gives you a known phenotype
find more resources at oneclass.com
find more resources at oneclass.com
- distance btwn 2 genes (ex. mutated gene and landmark gene) is described by gene linkage btwn them
- gene linkage – gene distance and
recombination fq (crossover fq)
- during meiosis (gametogenesis), diff genes will
be recombined to produce recombinants in the
next gen
- if genes are unlinked (ex. on diff chrm or far
apart on same chrm): recombinant type =
parental type, indep assortment
- if genes are linked (ex. close on same chrm):
genes tend to stay together in the gametes but
recomb can still happen due to cross-over
^ but recomb type will be less than parental
type in the next generations (more parental)
- rate of recomb is dep on the distance of genes, the closer the genes are, the less crossing over occurs btwn
- closer the genes are together wrt distance, the fewer recomb types in the next generations
- recomb fq is used to det the distance btwn mutated gene and a landmark gene in mapping/positional cloning
RF = # recomb / # of (parental + recomb)
^ RF expressed as %. 1% RF is known as a map unit (m.u) or centiMorgan (cM)
If RF = 50% or greater, genes are unlinked. If < 50%, two genes are linked. The lower the RF the close the genes
- traditionally, a mutated gene is mapped w visible (morphological) markers …
Ex. in drosophila, genes are seq – know the fn of many genes. If you mutate these known genes, get an easy to
detect phenotype. Can calculate the RF btwn your gene and a marker, to see if thee lose i the geoe
Ex. want to find which gene is mutated in the fly (short wing, mm)
^ dot ko hih h its o. In mapping line fly, all genes
except M are in their mutant form – known phenotype
- in F2, there will be combinations btwn the M gene and the W, B,
H and V genes in 4 diff chrms, the recomb fq btwn M and a given
landmark gene (W,B,H,V) can be det by analyzing the number of
recomb and parental types in the F2 progency
^ if RF = 50% + btwn the gene M and the landmark gene, M is
unlinked to that landmark gene on that chrm. If < 50%, the M gene is linked to landmark gene on that chrm
^ thus tells what chrm gene M is on bc you know what chrm the other genes are on (genome seq known)
- after identifying which chrm M gene is on, can try to narrow it down using other markers
- nowadays, a mutated gene is fq mapped w molecular markers
^ the traditional way involves many crosses – takes a long time.
- a ole ake is a sall DNA se polophiss hih usuall dot podue isile pheotpes
presented at the same chromosomal location btwn 2 inbred lines (strains) of species
- in Arabidopsis, Columbia-0, Col-0 and Landsberg (Ler) are 2 lines widely used for gene mapping. 99% the
same, but small diff btwn genomes, thus can use those different genes as the marker
- there are two types of molec markers widely used
1.SSLPs – simple seq length polymorphism markers
- often raised from varying numbers of repeats of simple sequences btwn line (ex.
Col-0 and ler)
- can be seen directly as bands after PCR on a gel ex. small vs big band
Ex. when mapped, in Ler, ciw12 marker in ler is slightly longer … thus a e used as a ake. The diff ot
gie ise to a pheotpe, ut ko its diff PC‘/gel eletophoesis shos diff ads… thus if ou also PC‘
w your plant, you can tell which line it is ex. LL or CC or CL
find more resources at oneclass.com
find more resources at oneclass.com
2.SNPs – single nucleotide polymorphisms
- sometimes can be detected by restriction enzymes after PCT as SNPS may either destroy of create a
restriction enzyme recognition size.
- SNP markers are often called cleaved amplified polymorphic seq markers (CAP)
- both frag are 200bp, but in Col-0, there is a single nt change within – leading to a
restriction site
- the ia PC‘, a tell hat lie plat is… if CC ill gie oigial seget s if LL
will give 1 (bc no restriction enzyme site)
Mapping a gene w molecular markers
- want to map where monster flower gene, in Ler Strain is located
^ cross w WT Col – given heterozygous F1. Then, self-cross to give recomb. Get F2 –
interested in mutant plant that shows the phenotype – in the locus of the mutate
gene it must be LL – bc only in Ler genome has LL
- in F2 population, there will be recomb btwn M and markers. In F2 plants
homozygous for monster flower, the genotype in the M gene is always LL when you
use ex. PCR to det the genotype of a given marker in F2 plants homozygous for monster flower
- if marker is LL, no recomb in 2 chrms... bc both mutant and marker come from
Ler
- if marker is LC, one recomb in 1 chrm… thee is oss-over during meiosis
- if marker is CC, two recomb in 2 chrms…^^
- then calculate the RF btwn the M gene and a given molec marker
Ex. mapping monster flower mutation in Ler. To map, cross w WT Col. F1,
self-cross, get F2. In F2, select mutant plant (genotype must be LL bc
mutant)
- linkage btwn monster flower and ciw12? (SSLP marker on chrm 1)
^ If control genome is Col, get cw12 band wrt CC. Get cw12 band in LL …
slightly larger than cw12 in Col, when using control Ler strain. Using LC
control, get 2 spec cw12 band ... correlating to CC or LL. (all controls)
- next, PCR w genomic DNA from the mutant plant. PCR to get band of
… # utat sas the stai is LL, # sas CC, ad # sas LL.
- after doing PCR on plants ex. 1-…. Get F utat plat esults
^ RF is multiplied by 2 on bottom by diploid and by 2 for CC on top btwn
recomb. RF btwn cw12 and monster flower gene is 52%, thus no
linkage btwn the genes…. Moste floe gee likel ot o h
- using nga 162 SSLP marker on chrm 3, map control strains and mutant plants w PCR
^ #1 is Col, #2 is Ler, #3 is Col etc. dep on how many F2 plants
* whenever you map w markers, need to genotype the control also
- RF is 8%, thus close linkage. Monster flower gene is likely on chrm 3
Why do we tend to use molecular markers not visible?
- with visible markers, you need to cross your mutant line to a mapping line where the landmark gene is
mutated to determine the RF btwn the mutated gene and a landmark gene, so many crosses are needed to
det the RF btwn the mutated gene and a given landmark gene (hard to have a mapping line where to many
genes are mutated)
- with molec markers, you only need to cross mutant line to WT of another strain once then you can det the RF
btwn the mutated gene and any given molecular marker, for ex. mutant strain ler (mm) x WT strain Col-0
MM… a the use PC‘ to hek the geotpe of akes at a loi i eah F poge hoozgous fo
mutant phenotypes
- many more molec markers can be found in the genome of a given species (great density of these akes…
small differences btwn genomes are common than having many diff known phenotypes
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Gene identification -> forward/reverse genetics in model species. Molecular cloning of a gene ->map-based cloning and confirmation of candidate gene. Study of gene expression: microarray, rt-pcr, northern, in situ, promotor-reporter gene fusion. Study of gene function: gene ko/knockdown to analyze gene function, rna interference, over and inappropriate expression of a gene. Protein purification: tag-based protein purification to quantify protein interaction in a complex. Protein characterization: sds-page and 2d-page for post translational modification. Genes are normally identified in a genetic model org. ^ easy to work with, grow fast, lots of progeny, small genome, most have sequence genomes, easy to be genetically manipulated to make transgenic lines, have international community of scientists working on same system thus can access more info. Arabidopsis thaliana is a model species for plant bio research: small in size 20cm mature, can grow indoors in a large number, short life cycle of 8-10 weeks, genome only have 140mbp, approx.
