Lecture 2 - Gene Analysis

Dr Federico Rojas
[email protected]
 Learning objectives

By the end of this lecture (+your own studying) you should be able to:
–Describe the steps involved in a typical agarose gel electrophoresis experiment for the separation of
DNA fragments
–Describe the stages of the polymerase chain reaction.
–Describe the stages involved in southern blotting analysis.
–Describe the basics of a typical DNA sequencing experiment.

Recommended Reading: Campbell and Reece 12th ed. Chapter 19, 452-454
 Agarose gel electrophoresis

• Nucleic acids are negatively charged (because of their phosphate backbone), so they migrate toward the
positive electrode.
• The gel acts like a sieve, selectively retarding the movement of larger molecules.
Visualizing DNA bands ultraviolet (UV) irradiation.

Ethidium bromide fluoresces under

UV light
Visualizing DNA bands in an agarose gel by EtBr staining and ultraviolet (UV) irradiation
 Agarose percentage

The composition of the gel determines the sizes of the DNA molecules that can be separated.

A 0.5 cm-thick slab of 0.5% agarose, which has

relatively large pores, would be used for
molecules in the size range 1 to 30 kb .

Agarose % (w/v) Resolution

0.50 % 1,000 – 30,000 bp
0.70 % 800 – 12,000 bp
1.00 % 500 – 10,000 bp
1.20 % 400 – 7,000 bp
1.50 % 200 – 3,000 bp
2.00 % 50 – 2,000 bp
Techniques to study gene/DNA structure (sequence)

1. Restriction Mapping/Restriction
2. Fragment Length Polymorphism (RFLP)
3. Polymerase Chain Reaction
4. Southern Analysis
5. DNA Sequencing
 Restriction mapping
Mapping the positions of different restriction sites in a DNA molecule

To construct a restriction map, a series of

restriction digests must be performed.

1. The number and sizes of the fragments

produced by each restriction endonuclease must
be determined by gel electrophoresis.

2. Information must then be supplemented by a

series of double digestions (two restriction
endonucleases at once)
 Marker XbaI XhoI XhoI+XbaI

Comparing the results of single and double digests will allow many,
if not all, of the restriction sites to be mapped
33-

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The only possibility is:

 Restriction mapping - Estimation of the sizes of DNA molecules
Gel electrophoresis separates different-sized DNA molecules, with the smallest molecules
travelling the greatest distance toward the positive electrode.
Restriction fragment length polymorphisms (RFLP) analysis
An RFLP is a sequence variation that changes a restriction site

Human breast cancer susceptibility gene

BRCA1: a significant number of the women
who suffered from the disease all possessed
the same version of an RFLP called D17S74.
 Polymerase chain reaction (PCR)

Kary Mullis invented the polymerase chain reaction (PCR)

 Thermostable DNA polymerases

In the 1960s, Thomas Brock was a biologist at

Indiana University.

He started a field research station in Yellowstone

National Park.

At the time, scientists believed that bacteria optimally

lived at about 55°C, and that nothing lived above
73°C

The bacteria: Thermus aquaticus, which contains the DNA polymerase that ultimately
became the backbone of PCR: Taq Polymerase
 Polymerase chain reaction (PCR) - 1

PCR uses repeated rounds of strand separation, hybridization, and synthesis to amplify DNA

1. Denaturation DNA at 94°C 2. Annealing at 50–60°C 3. Synthesis of new DNA at 74°C

Molecular Biology of the Cell, 6th edition

Polymerase chain reaction (PCR) - 2

Repeat the cycle 25–30 times Molecular Biology of the Cell, 6th edition
 Polymerase chain reaction (PCR) - 3
• Because a heat-resistant polymerase is used, the reaction can be repeated continuously without
the addition of more enzyme.
• Each cycle doubles the copy number of the amplified gene:
• 10 cycles ideally produce: 1,024 (210) copies
• 30 cycles yield (210x3) = 109-fold amplification
 PCR uses

• Amplification of the desired sequence prior to cloning

• Diagnostic and Forensic applications
• Species identification (meat scandals)
• Identification of disease alleles
• Gene expression (next lecture)

• Sensitivity – theoretically only one target molecule required

• Specificity – The polymerase chain reaction allows to obtain a pure sample of a gene.
 Southern blotting

Allows specific fragments to be identified in a complex mixture

Named after Ed Southern.

As well as colony hybridisation analysis, there are also occasions when it is

necessary to use hybridisation probing to identify which of a series of restriction
fragments contains a gene of interest

B = BamHI restriction site.

Southern blotting - 2

The DNA to be analysed is digested with a

restriction endonuclease, and the digested DNA
fragments are separated by gel electrophoresis.
 Southern blotting (continue)

The same method can also be used for the transfer of:
RNA molecules (Northern transfer) or
Proteins (Western transfer)

So far, no one has devised Eastern transfers.

Nucleic acids are generally detected by hybridisation with homologous sequences.

renature to form
double-stranded
molecules
 Frederick Sanger

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 Sanger sequencing
Each of the four dideoxynucleotides is labelled with a different fluorescent dye, so their incorporation into
DNA can be monitored.

The incorporation of a dideoxynucleotide stops further DNA synthesis because no 3′ hydroxyl group is
available for the addition of the next nucleotide.

X
 Sanger sequencing - 2

Thus a series of labelled DNA molecules is generated, each terminating at the base represented by
a specific fluorescent dideoxynucleotide.
Possible to obtain over 750 bp of sequence per experiment.
Genes are longer than this: carry out two or more chain-termination experiments, directed at
different parts of a gene, in order to build up the complete sequence.
 The chain-termination (Sanger) method was also used to obtain the first complete genome sequences.

Challenging 3200 Mbases

Furthermore, no sequencing method is entirely accurate it is necessary to sequence each region of a

genome multiple times, in order to identify errors present in individual sequence reads
With the chain-termination method, at least a fivefold sequence depth or coverage is required, which
means that every nucleotide is present in five different reads.
 Cycle Sequencing
The key difference with traditional Sanger method: the employment of a thermo stable DNA
polymerase.
The advantage: the sequencing reaction can be repeated over and over again in the same tube by
heating the mixture to denature the DNA and then allowing it to cool down to anneal the primers
and polymerise new strands.

Therefore less template DNA is needed than for conventional sequencing reactions.
 Lecture 2 - Gene Analysis

By the end of today (+your own studying) you should be able to:

–Describe the steps involved in a typical agarose gel electrophoresis experiment for the
separation of DNA fragments
–Describe the stages of the polymerase chain reaction.
–Describe the stages involved in Southern blotting analysis.
–Describe the basics of a typical DNA sequencing experiment.