Section 4Lesson 5– Human

Therapeutics

PCR Quiz1. What is special about Taq?

2. What temperature is best for the synthesis stage in the process?

3. Give 2 possible applications of this technique.

4. What do primers do?

5. Which way does Taq transcribe (3’ – 5’ OR 5’ – 3’)?

It is a thermal stable enzyme able to operate at higher temperatures.

72˚C

DNA sequencing, diagnosis of hereditary illnesses, diagnosis of infectious diseases, phylogeny, genetic fingerprinting

Anneal to target sequences

3’ – 5’

Dideoxy chain-termination Method

This is sometimes know as the Sanger method as it was first developed in 1977 by Frederick Sanger in Cambridge.

Sanger sequencing is modelled after the natural process of DNA replication, and it uses ‘dummy’ nucleotides to stop replication whenever a specific nucleotide is encountered. Because this truncated replication occurs over and over again, nucleic acids of varying lengths accumulate and can be used to determine the position of each nucleotide in the sequence.

How Does it Work?

Sanger Method Video

Sanger Sequencing

How Does it Work?1. PCR is used to amplify the section of DNA that is to be sequenced.

2. The DNA is then heated to denature it and a primer is added that corresponds to the start of the sequence.

3. DNA polymerase dNTPs are also added. These are deoxynucleotide triphosphates, or dNTPs (where the "N" is a placeholder for A, T, G, or C). These will create a normal copy of the strand. Crucially at this stage dideoxynucleotide triphosphates, or ddNTPs are also added. These are chemically slightly different (lacking an –OH group). This difference prevents them from binding to the next dNTP (or ddNTP) and caused transcription to STOP. These were radioactively labelled so they could be spotted on the gel at the end. Originally 4 flasks were set up – each with a different ddNTP added. Now this is done in a single flask.

4. The result is a mixture of strands of varying lengths each ending in a terminal ddNTP that indicates whether it is A,T,G or C.

5. Gel electrophoresis is used to separate out the different lengths of DNA.

deoxyribonucleoside triphosphate

dideoxyribonucleoside triphosphate

Allows strand extension at 3’ end

Prevents strand extension at 3’ endIncorporation of this nucleotide results in chain termination.

DNA fragments can be separated by gel electrophoresis

DNA moves to the positive terminaldue to it’s overallnegative chargegel with DNA

fragments

+

Smallest fragments

Largest fragments

Separating DNA Fragments

Gel Electrophoresis

Reading a Gel

Use the handout of the gel to read the

sequence of this DNA fragment.

Automated MethodThe original method worked well but was time consuming. It was later adapted to use fluorescent labelling instead of radioactive labelling.

This method allowed the labels to be detected as they ran out the end of the gel in the correct order.

Next Generation Sequencing

Wiki - next generation info table

Comparative Genome Analysis

As our knowledge and understanding of individual genomes has increased, our ability to make comparisons between genomes has also increased.The monograph tells us that in general, the more complex the organism is the more complex the genes are and the more genes are present. More recent research has proven this to not always be the case but that further comparative analysis is needed.

Organism Genome Size (Mb)

Number of Genes

E. coli(bacteria)

4.6 4,405

Saccaromyces cerevisiae(yeast)

12.1 5,800

Drosophila melanogaster(fruit fly)

150 12,000 (15,000 2013)

Homo sapiens(human)

3,000 70,000(22,000 2013)

Nicotiana tabacum(tobacco)

4,500(3,000 2013)

43,000

Your Tasks1.Add the red terms to your glossary.2.Past Paper Questions due Monday

March 4th.

2005 MC Q11,122006 MC Q 122007 Q72008 MC Q 10,11,122009 MC Q 12, ER Q8B2010 MC Q 11,12,13