Probes, DNA amplification and genomic libraries

Question 1

Detection of a specific gene

Answer 1

(e. g. Amigo 2 in brain sections)
1. labelled mRNA coding for the Amigo2 protein
2. Chemically synthesised DNA matching part of the Amigo2 gene (oligonucleotide)

Question 2

What are the PCR minimum requirement?

Answer 2

• Minimum requirement:
– template DNA
– DNA polymerase (in a compatible buffer)
– primers* *means that ends of the DNA template must be known
– dNTPs

Question 3

What is the Problem in PCR? and what is the solution?

- Temperature

Answer 3

Above the Tm, DNA polymerase is rapidly inactivated AND primers will not anneal
• Solution:
– Use of a thermostable polymerase to allow >20 cycles without inactivation
– Temperature cycling to allow primer binding (annealing temperature) and optimum DNA
polymerase activity (extension temperature)

Question 4

What is Taq polymerase?

Answer 4

Made possible by the discovery of bacteria that live in extreme heat and isolation of their DNA polymerase.
Bacterium is thermophilic,
Thermus aquaticus – giving us
Taq polymerase

Question 5

How can PCR be used for Altering DNA sequence (site-directed mutagenesis)?

Answer 5

-During PCR, primers become incorporated into the
product
-If primer contains a change (mutant oligonucleotide),
site-specific mutation occurs
-Used to introduce amino acid changes, restriction
sites and changing regulatory sequences

Question 6

What are the Uses of PCR

Answer 6

-Altering DNA sequence (site-directed mutagenesis)
-Amplify DNA for cloning purposes -> Produce many copies of a gene by amplification
-Amplify DNA to detect a sequence->Produces a positive signal if a specific sequence is
present in the sample
-Obtain genomic or cDNA clones ->PCR can be used as a probe to fish out genomic or
cDNA clones
Short clones can be amplified directly using the
appropriate primers

Question 7

what is qPCR and what it can be used for:

Answer 7

qPCR stands for quantitative polymerase chain reaction and is a technology used for measuring DNA using PCR

• Gene expression profiling
• Copy number variation
• Molecular diagnostics
• Genotyping

Question 8

How can PCR be used in genetic testing?

Answer 8

•Screening for breast cancer genes
• BRCA1 and BRCA2 produce tumor suppressor proteins
• Inherited mutations where BRCA proteins are not
produced –sometimes results in cancer

Question 9

How can PCR be used for Paternity testing?

Answer 9

• In the human genome, there are many short, repeated sequences that are heritable from your mother and father
• One example is the short tandem repeat (STR) that can easily be amplified using PCR
• Common approach in paternity testing cases
• Children inherit half their alleles from each parent
– should possess a combination of parents alleles

Question 10

PCR in forensic science

Answer 10

• DNA repeats known as VNTR (variable number
of tandem repeats) are highly variable in the population
• Typically longer than STRs - up to 100bp
• Individuals inherit each VNTR locus from their
mother and father
• A pair of primers that bracket this VNTR loci
will provide bands of different sizes as inherited from each parent
• PCR useful as minute starting material is sufficient

Question 11

Using PCR in forensic science

Answer 11

Short repeated sequences of variable length useful for
identification
• Known as VNTR (variable number of tandem repeat)
• Individuals usually inherit a different variant from each parent
• Two unrelated individuals usually do not share same pair of sequences
• PCR analysis using primers to bracket the locus results in PCR products that can be viewed in
the gel

Question 12

poly-A tail

Answer 12

poly-A tail is used as a specific marker distinguishing mRNAs from other RNAs
(e.g. rRNAs and tRNAs)
same DNA in most cells. Different phenotypes result from variations in gene expression and protein
activity

Question 13

Gene regulation

Levels of mRNAs differ for:

Answer 13

• different genes in the same cell
• the same gene in different cell types or differentiation states
• the same gene in response to external signals
(e.g. viral infection, insulin levels, oxidative stress…)

Question 14

Full Genomic library amplification

Answer 14

Contains the entire genome of an organism
• Naturally occurring F plasmids can accommodate
DNA fragments of 300,000 to 1 million
nucleotide pairs
• F plasmid or its derivative called Bacterial
artificial chromosomes (BAC) is present only in
one or two copies per E.coli cell
• BACs are now the preferred vector for making
DNA libraries

Question 15

What is a Genomic DNA library?

Answer 15

• Larger fragments can be inserted into BACs
• Each plasmid carries a portion of the genome
• The entire collection of the plasmid carrying a representation of the whole genome is called the genomic DNA library

Question 16

limitation/cons of genomic library

Answer 16

• Genomic DNA clones obtained from DNA of higher
eukaryotes contain non-coding DNA (introns, satellite
DNA etc.)
– Much larger fragments, some of which do not contain genes
– Not directly usable for protein expression
• Genomic clones are the same for a given organism
irrespective of the cell type or specific condition
– Not representative of the transcriptome (i.e. part of the DNA that is expressed in a particular condition in a particular cell)

Question 17

cDNA libraries

Answer 17

• An alternative is to select only DNA sequences that are transcribed into mRNA and should correspond to protein-coding genes
• Done by extracting mRNA and reverse transcribing into DNA making complementary DNA or cDNA
• Single-stranded cDNA molecules converted into double stranded molecules with DNA polymerase
• cDNA inserted into plasmid and cloned
• Entire collection of clones makes a cDNA library
• cDNA clones contain only regions of the genome that have been transcribed into mRNA (= the transcriptome)
• Different tissues produce distinct sets of mRNA molecules, so distinct cDNA libraries are obtained for each type of cell used

Question 18

cDNA synthesis

Answer 18

• Poly T hybridised to poly A tail of mRNA to act as a
primer for reverse transcriptase
• RNase H makes nicks and gaps in RNA
• DNA polymerase copies single stranded DNA into
double stranded cDNA
• cDNA clones contain only the transcriptome
• Distinct cDNA libraries are obtained for each type of
cell used

Question 19

Differences between cDNA and genomic DNA clones

Answer 19

• Genomic library contains exons, introns and
untranscribed regions
– exhaustive
– used for sequencing
– untranscribed DNA is used in some techniques (e.g.
RFLP)
• cDNA library contains only transcribed genes
– provides information about the identity and
quantity of each transcript

Question 20

what is microarrays and two different types of it?

Answer 20

Microarray is prepared by attachment or synthesis of
DNA probes
– spotted arrays: attachment of probes prepared separately
– oligonucleotide arrays: in situsynthesis of oligonucleotide probes
• Genome chips (gene arrays) may have >5 million probes
– designed for a specific application (human vs. mouse; disease-specific; bacterial genotyping…)

Question 21

how microarrays is used for the analysis of gene expression?

Answer 21

• DNA microarrays analyse gene expression by monitoring mRNA products of thousands of genes at
once;
• Convert mRNA from cell being studied into cDNA which is then labelled with a fluorescent probe;
• Labelled probe is hybridised to microarray;
• Scanned to see which genes the
probe binds to;
• Reference probe used to give relative
expression

Question 22

Using microarrays for allelic variation screening

Answer 22

• DNA microarray to detect polymorphisms within a
population (e.g. SNPs)
• Hybridise DNA of interest
• Array contains target sequences – genotyping
microarray containing all disease-associated variants in
candidate genes

Question 23

what are short tendem repeats ?

Answer 23

Short tandem repeats (STRs), which are sometimes referred to as microsatellites or simple sequence repeats (SSRs), are accordion-like stretches of DNA containing core repeat units of between two and seven nucleotides in length that are tandemly repeated from approximately a half dozen to several dozen times

Question 24

What is Gene expression profiling?

Answer 24

Gene expression profiling is the determination of the pattern of genes expressed, at the level of transcription, under specific circumstances or in a specific cell to give a global picture of cellular function