L4 - Use of PCR

Question 1

What can PCR amplify DNA for?

Answer 1

Genetic testing 
Diagnosis 
Personalised medicine 
Forensics 
Measuring RNA levels by RTPCR
Manipulating DNA

Question 2

What is RTPCR?

Answer 2

Reverse Transcriptase PCR

A variation of the polymerase chain reaction that typically measures RNA expression levels. In RT-PCR, complementary DNA (cDNA) is made by reverse transcribing of the RNA templates with the enzyme reverse transciptase

Question 3

How can using PCR to create novel DNA molecules help us to better understand life?

Answer 3

Helps us to understand:
• Protein function & structure
• Gene function
• Protein localisation

Question 4

Why is there no PCR for proteins?

Answer 4

It is not possible to make protein with a single enzyme

Cell components needed

Need transcription & translation

Question 5

How to make proteins for PCR products

Answer 5

Clone polymerase alpha using tagging primers to introduce Nde1 & BamH1 restriction enzyme sites

Primers to introduce artificial Nde1 (CATATG) & BamH1 (GGATCC) sites to Pol alpha DNA, with 6bp spacers

Pol alpha primers to introduce Nde1 & BamH1 sites

Cut PCR product with Nde1 & BamH1, ligate into pET16b (vector), cut with same enzymes

Culture E.coli with plasmids

Induce expression of PCR insert via T7 RNA polymerase – 2 hours

Extract proteins & purify proteins

Question 6

How to transform (transfect/introduce) artificial PCR fragment into yeast genome

Answer 6

1. Use hybrid primers to amplify a drug resistance cassette from plasmid
2. Introduce appropriate yeast DNA sequences at each end of PCR product
3. Transform PCR product into yeast
4. Yeast ‘repair’ the ‘broken’ fragment by homologous recombination using ends of PCR prduct
5. If DNA inserts into genome, cells become resistant to a drug

Question 7

What can you learn from organisms with altered genomes?

Answer 7

• Effects of deleting gene?
• Alive or dead?
• Sensitive to DNA damage?
• Able to grow under all conditions?
• Susceptible to disease?

Question 8

How can you use GFP to determine fusion proteins?

Answer 8

GFP tag (238 aa) is analogous to polyhistadine tag – fuse to protein

Integrate into genome

Allows fusion protein location to be determined in live organisms