Analysis of nucleic acids

Question 1

How is DNA cloned in-vivo?

Answer 1

Selective propagation of certain agar colonies by transforming it onto host cells and then eliminating those not wanted.

Question 2

How is DNA cloned in-vitro?

Answer 2

1. Cutting target DNA and replicon - plasmid with SAME restriction endonuclease so they can join
2. Mixing and joining the two with DNA Ligase

Question 3

What are restriction endonucleases and how do they function?

Answer 3

Enzymes that cleave DNA at specific sequences, leaving blunt or sticky ends.

Recognises PALINDROMIC sequences

`is a primitive immune system as host DNA is methylated and hence cant be broken

Question 4

How is DNA separated?

Answer 4

Electrophoresis
- smaller fragments go further toward anode - positive as DNA is negative

DNA can then be isolated by hybridisation

Question 5

What is nucleic acid hybridisation?

Answer 5

The process of detecting specific nucleic acid sequences by using a probe to identify its complimetary strand.

Target DNa is immobilised on nylon support, and probes added
These probes join and are fluorescent.

Forms of hybridisation
Southern - DNA on DNA target
Northern - DNA on RNA target

Question 6

How are DNA probes denatured?

Answer 6

Heating to break H bonds

Depend on

1. Strand length
2. Base composition
3. Chemical environment

Question 7

What does melting temperature indicate? And what is hybridisation stringency

Answer 7

Indicates duplex stability

HS is ability to distinguish between related sequences
- Increases with temperature and decrease in [Na+]

Question 8

What is PCR?

Answer 8

In vitro method of amplifying specific target DNA

Question 9

How is PCR carried out?

Answer 9

2 primers made - for each direction, complementary to the DNA needed to be copied

Added with dNTPs and TAQ polymerase to mixture

1. Denature - split the DNA so primers can join
2. Anneal - primers join
3. Extend - TAQ polymerase allows dNTPs to be added and hence make new strands.

Question 10

How are primers designed?

Answer 10

20 nucleotides long

No tandem repeats

No complementarity to avoid dimers

Question 11

What are the applications for PCR?

Answer 11

DNA sequencing

cDNA cloning

Detecting point mutations

Question 12

What is a DNA microarray?

Answer 12

Collection of microscopic DNA representing individual genes

Used to monitor expression levels of genes - dual profiling