Southern blotting, DNA sequencing Flashcards
What is meant by a DNA molecule denaturing?
Hydrogen bonds in base pairs are broken
DNA becomes single-stranded
How is DNA denatured?
Heating it to high temperatures
Treated it with alkaline solution
What is meant by DNA renaturing?
Hydrogen bonds in base pairs reform
form double-stranded DNA
How is DNA renatured?
Cooling it down
What is DNA hybridisation?
Add labelled DNA probe to single-stranded DNA that has complementary base sequence
DNA probe will bind to DNA
What is a DNA probe?
Short single-stranded section of DNA
How can DNA probes be labelled?
Radioactively
Fluorescent markers
What is the purpose of DNA probes being labelled?
So can identify them bound to DNA
What is the first step of Southern blotting?
DNA gel electrophoresis
What is done to DNA gel electrophoresis results in Southern blotting?
Gel is soaked in alkaline solution
to denature DNA
What is done to the denatured DNA on the gel electrophoresis results in Southerin blotting?
Single-stranded DNA fragments are moved to nitrocellulose paper by capillary action
What is done to the nitrocellulose paper in Southern blotting?
Placed in solution containing labelled DNA probes
then washed off to remove unbound DNA probes
What is done to identify the presence of DNA probes on the nitrocellulose paper?
Use photographic film if DNA probes are radioactively labelled
Fluorescent detection methods if DNA probes are fluorescently labelled
Do DNA probes have a completely complementary base sequence to the target DNA sequence?
No
What affects how tightly DNA probes bind to the target DNA sequence?
How complementary the DNA probes are to the DNA sequence - the more complementary, the more tightly they bind
Do DNA probes have to completely align with the DNA target sequence?
No, only part of them has to bind
What is DNA sequencing?
Process of working out base sequence of a DNA fragment
How is a ddNTP different to a dNTP?
dNTP 3’ is bound to OH
ddNTP 3’ is bound it H only
How does a ddNTP affect elongation of a DNA strand? Why?
DNA polymerase cannot add next nucleotide due to lack of 3’ OH
so elongation of DNA strand is terminated
What are the requirements of DNA sequencing?
Four containers, each with
- DNA fragment to be sequenced
- DNA primer
- dNTPs
- DNA polymerase
- one type of ddNTP
What is a condition of the DNA primer in DNA sequencing? Why?
Must have complementary base sequence to 3’ end of DNA fragment
since DNA polymerase will extend new DNA strand 5’ to 3’
Does DNA polymerase add on a dNTP or a ddNTP to the new DNA strand in DNA sequencing?
Chance of either!
What is done to the contents of the four containers after they’re incubated?
For each container, new DNA strands put into a lane
then DNA strands separated out by DNA gel electrophoresis
How are the results of DNA sequencing interpreted?
Read off sequence by furthest band to closest band
complementary base sequence will be that of DNA fragment
