DNA 1 - Toolkit techniques

Question 1

Restriction Nucleases

Answer 1

Particular enzymes
They stop incoming infection ( particularly viral infections )
They have the ability to cleve nucleotides –> chopping them up very small until they become inactive

There are different types of restriction nucleases that cut different organisms and DNA

Question 2

Two ways restruction nucleases cut DNA

Answer 2

1. Clean Break

2. Break in an asymetrical fashion –> creating and overhang.

Question 3

How to restriction nucleases know where to cut

Answer 3

Controlled by the sequence. The proteins recognise particular portions (particular sequence ) within DNA to cut it

Question 4

What is the self recognition feature

Answer 4

This prevents the bacteria from breaking down its own DNA

Mediated by methylation in their own DNA that serves as a way of self DNA –> When DNA doesn’t have this the restriction nucleases act

Question 5

Electrphoresis

Answer 5

Understand how this works

- Can be used to develop restriction maps –>After enzyme incubation , the different DNA sizes can be compared

Question 6

Restriction Maps

Answer 6

The map depends on the DNA sequence and therefor can shoe differences between similar DNAs
The ultimate map depends on the complete nucleotide sequence

• Used for forensic profiling
• Used for paternity testing

Question 7

Sangha sequencing method

Answer 7

-Take double stranded DNA
- Heat it to make the double stranded DNA single stranded
- Add a labelled primer ( a short sequence of DNA that is complementary to the single stranded DNA molecule of interest)–> So you have complimentary base pairing
- Start adding nucleotides and DNA polymerase for filling in nucleotides for the complementary section
-Also had dideoxyribonucleotides (these differ from deoxyribonucleiotide because the 3’ hydroxyle group in the deoxyribonucleotides are replaced by a hydrogen group)This stops strand extension.
- Single stranded DNA molecule +primer +ATP.
-Then wash out
Then add TTP.

• Seperate fragments on a gel–> from 5’ to 3’

Question 8

Nucleic Acid Hybridisation

Answer 8

Once seperated by size
We don’t know which gene is present in each fragment.
To find this out we use nucleic acid hybridisation
- Strands heated to seperate
- If the strands are cooled slowly reannealing or hybridisation can occur
- Hybridisation allows you to search for a DNA sequence of interest
- A DNA probe is a short single stranded DNA which is used to detect complementary sequences ( this is complementary to the gene of interest so will anneal onto the gene of interest) Normally has a signal built into the probe which means we can detect when they have attached

• Used for pre natal diagnosis or genetic disease

Question 9

Southern blotting

Northern blotting

Answer 9

Check these

Question 10

In Situ Hybridisation

Answer 10

Hybridisation on whole cells
Can detect DNA in chromosomes or RNA in cells
Fluorescently labelled DNA can hybridise to whole chromosomes briefly exposed to high PH

Can also be used to detect mRNA –> helps understand which parts of a tissue preferentially express mRNA
Used in studies on embryo development

Question 11

Southern Blotting

Starting material?

Answer 11

DNA

Question 12

Western Blotting

Starting material?

Answer 12

Proteins

Question 13

Northern Blotting

Starting material?

Answer 13

RNA