LSM2254 Fundamentals of Plant Biology

Practical 4:

Identification of a T-DNA insertional mutant in

Arabidopsis (II)

Dr. Lau On Sun

Department of Biological Sciences

onsunlau@nus.edu.sg

21 March, 2021

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Practical 3 & 4

Overall objective
• Determine the genotype of hy5q, a T-DNA insertional
mutant of HY5

Objectives for Practical 4

• Analyze PCR samples amplified from the DNA of wild-
type, hy5q (Practical 3) and an unknown plant by
agarose gel electrophoresis

• Confirm and determine the genotype of the plants

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How do we know we are working on the correct mutants?

hy5q
WT

• We can look at the gene locus and check if there is a mutation

• Since hy5q is a T-DNA insertional mutant, confirmation can be done

by examining if a T-DNA is present on the HY5 locus

x
HY5
locus:
WT T-DNA mutant
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Determining the genotype of a T-DNA mutant (more in P4)
At the At the
HY5 LP HY5 TP
locus: locus:

RP T-DNA RP

LP and RP primers:

Specific to HY5 locus

TP primer:

hy5q Specific to the T-DNA

WT

• The gene-specific insertion of the T-DNA can be detected by PCR

• To determine the presence/absence and the zygosity of the insertion,

3 primers are used for 2 PCR reactions

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Type of genotypes of a putative T-DNA mutant

• Arabidopsis is diploid, i.e. have two sets of chromosomes

WT

e.g. HY5+/+ + +
T-DNA

Heterozygous

e.g. hy5+/- + -

Homozygous

e.g. hy5-/-
- -

How does our 3 primers, i.e. LP, RP & TP,

help in distinguishing these cases?
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Detecting gene-specific insertion of the T-DNA by PCR
In WT does not bind to WT DNA
TP
LP
HY5
locus:
RP

LP+RP mix: TP+RP mix:

LP

TP does not bind to WT

RP No PCR product
PCR product
In our experiment:

the amplified target of the HY5

gene is ~1150 bp

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Detecting gene-specific insertion of the T-DNA by PCR (cont.)

In homozygous hy5q (hy5-/-)

LP TP
HY5
locus:
RP
T-DNA

LP+RP mix: TP+RP mix:

LP and RP still bind to the HY5 sites;
TP
BUT, the T-DNA is rather long (~5000
bp)
RP
Our PCR conditions do not allow good PCR product
amplification of this size (~6000 bp)
In our experiment:

No PCR product (and definitely no the amplified target of the

band with a size of 1150 bp) T-DNA-HY5 hybrid is ~700 bp

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Detecting gene-specific insertion of the T-DNA by PCR (cont.)

In heterozygous hy5q (+/-)

TP
LP
HY5
locus:
RP
LP TP
HY5
locus:
RP
T-DNA

What will be the results of LP-RP and TP-RP reactions?

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Previously… Plant DNA extraction and PCR reactions

Plant DNA Quantitate and

extraction set up PCR

Plant samples Purified genomic

DNA 2 genotypes 2 PCR mixes
WT
LP-RP (pink)

x
hy5q TP-RP (blue)

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Today: Cast and run gel and analyze PCR products

1. Cast gel

• 1% TAE gel with 8 wells

2. Prepare and run samples

• 6 samples and the DNA ladder

• 2 PCR products (LP-RP and TP-RP reactions) each of

• WT, hy5q (use 10 µl) and an unknown sample (Uk) (use 5 µl)

3. Gel documentation

• Identify the bands in the samples

• Analyze results and confirm/determine genotypes of the plants

• Keep a record of the gel picture for the lab assignment

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