Background

• Genetic disorders are often the result of gene mutations.

• People with a mutant allele often have a family history of the disease.

• It is important they are screened for the disease to help determine the likelihood of their offspring inheriting the disease.

Background continued

• We can use DNA probes to locate specific genes but in order to do this we need to know the sequence of bases in the gene we are screening for (as the probe will be complimentary to some of these bases)

• Read the middle of page 13 – Medical Diagnosis.

DNA probes

• Are a short single stranded piece of DNA which are ‘labelled’ (they are either radioactive or fluorescent). They are complementary to a portion of the gene and enable the gene to be visualised.

• There are 2 main types of label used:

Radioactive labels:

• These are made using nucleotides which contain radioactive phosphorus.

• They can be identified using a photographic plate.

Do you remember the principles of gel electrophoresis?

Fluorescent labels:

• These emit light (fluoresce) under certain conditions.

Aims

• To know what a DNA probe is.

• To be able to work out the sequence of bases in a plasmid using restriction mapping.

• To be able to work out the sequence of bases in a gene using gel electrophoresis.

Restriction mapping to determine the base sequence of

a linear gene

• Most genes are too long to be sequenced in one go so are cut into smaller segments using restriction enzymes first.

• Each section is then sequenced and then restriction mapping is used to put them in the right order thus giving the overall base sequence of the gene.

• Before we try this with linear genes we will have a look at restiction mapping in plasmids. (pg 14 booklet)

Restriction Mapping

• Restriction endonuclease enzymes can be used to cut a long piece of DNA into shorter segments which can then be sequenced.

• These shorter segments must then be put in the correct order to give the sequence of the original long piece of DNA.

• This is restriction mapping.

• Draw a plasmid on your whiteboard. Then answer the questions below:

1.How many pieces of DNA would be produced if a restriction enzyme was used to make a single cut in your plasmid?

2.How many pieces of DNA would be produced if 2 enzymes were used to cut the plasmid?

Basic idea:• Making one “cut” in the plasmid, using only

one restriction enzyme, results in one linear piece of DNA

• Making two “cuts” by using two restriction enzymes will give 2 pieces of DNA of different lengths (probably).

• Using different combinations of two restriction enzymes then comparing the fragments produced using electrophoresis you can work out the sequence of the fragments within the plasmid i.e. restriction mapping.

• We will now work through the example on page 14 together.

http://www.youtube.com/watch?v=v2T8Y3-8674&feature=related

Now try this one:Pairs of restriction endonucleases used

Hind III and BamH1

Hind III and Not1

Not1and BamH1

100 -

60 -

50 -

40 -

30 -

20 -

10 -

90 -

80 -

70 -

• By combining this with DNA sequencing a long piece of DNA can be cut by restriction enzymes into shorter more manageable segments that can then be sequenced to give the order of bases in the original longer piece of DNA.

Complete the Application exercise on page 270 of your text book

• Now try the exercises on the small sheet

Restriction mapping to determine the base sequence of

a linear gene

• Most genes are too long to be sequenced in one go so are cut into smaller segments using restriction enzymes first. Each section is then sequenced and then restriction mapping is used to put them in the right order thus giving the overall base sequence of the gene.

• http://www.youtube.com/watch?v=WX8V1SWQbFw

Total digest Partial digest

HindIII digest

Eco R1 digest

Radioactive fragments (from total digest)

10kb

8kb

5kb

3kb

2kb

1. Use the total digest to state how long the gene is?

2. Can you explain the result for Eco R1?

3. How many times does Hind III cut the gene?

4. What is the length of the fragment at the beginning of the gene

5. Use the information to construct a restriction map for the gene.

DNA sequencing

• This also uses the “idea” that DNA strands separate when heated and can be used to work out the sequence of bases in a gene.

• http://www.huffingtonpost.com/2014/03/22/largest-genome-sequenced-loblolly_n_5008965.html

• In order to find out the sequence of a section of DNA, the following need to be used:

COMPONENT ADDED REASON

DNA sample to be sequenced (single stranded)

This is the sequence we are trying to find out. It needs to be single stranded so complementary nucleotides can attach to it

Nucleotides To make the complementary strand

Terminator nucleotides (either A, T, C or G)

To provide different sizes of fragments to be identified

Labelled primer/probe (radioactive or fluorescent)

To start replication and enable the fragments to be visualised

DNA polymerase To join nucleotides forming a complementary strand

Are you ready to sequence your first gene?

Firstly obtain your single stranded DNA using restriction enzymes then add a probe to the starting sequence.

Put the single strand of DNA to be sequenced in a test tube and add all 4 normal nucleotides and one terminator nucleotide (here terminator C).

ATCGACCGTAGACT Unknown DNA sample

Labelled primer attaches to start sequencing

ATCGACCGTAGACT

DNA polymerase starts to add complementary nucleotides

ATCGACCGTAGACT

This continues until a terminator nucleotide is added (in this case cytosine)

Write down all the fragments which could be produced in the terminator

C test tube.

• TAGC

• TAGCTGGC

• TAGCTGGCATC

Write down all the fragments which could be produced in the terminator

T test tube.

• T

• TAGCT

• TAGCTGGCAT

• TAGCTGGCATCT

• Now complete the sequencing exercise on page 18

• Now use the worksheet to sequence your first gene.

More Sequencing in the news

• http://www.bbc.co.uk/news/health-20314024

Medical diagnosis

• Genetic disorders are often the result of gene mutations.

• People with a mutant allele often have a family history of the disease.

• It is important they are screened for the disease to help determine the likelihood of their offspring inheriting the disease.

• Read page 271 – 273 now and complete page 32 of your booklet including the application.