analysing DNA and RNA by hybridisation and sequencing

a map of DNA genomes can be generated using what two mechanisms?

• restriction enzyme digest

• agarose gel electrophoresis

why is it easier to map out small genomes than larger ones?

• Digestion of larger more complex genomes will create thousands of fragments differing by size and many of the same size 

• Appears as a smear on the agarose gel -> many DNA fragments migrating very close together

• Harder to distinguish DNA fragments

how can we identify a specific DNA fragment in the complex mixture?

by hybridising a labelled probe to the DNA fragment

hybridisation

• Combining different single strands/fragments of DNA/RNA together

• Used to detect specific DNA/RNA fragments in agarose gels

hybridisation can be used to synthesise (?)-stranded DNA

double

probes

• single stranded labelled DNA

• hybridised to single stranded DNA or RNA

why are high temperatures needed for hybridisation?

• to ensure only complementary strands completely anneal together

• when the temperature isn't high enough, non-complementary strands can hybridise

• loops can form in regions when temp isn’t high enough

name two types of hybridisation techniques

1. southern blotting → DNA

2. northern blotting → RNA

DNA vs RNA gel electrophoresis

1. DNA

• Isolate DNA from cells and digest with a restriction enzyme

• Agarose gel electrophoresis → separate using 0.5% - 2.0% agarose gel

• Negatively charged DNA migrates towards positive electrode

• Separated by size -> migrates as double stranded DNA

 

2. RNA

• Isolate RNA from cells -> RNA molecules don't need to be digested

• Agarose gel electrophoresis → separate using 1%-1.5% agarose gel

• Denaturing agent added to agarose gel -> removes secondary structures from RNA (structures that base pair with itself)

• Ensures that RNA migrates as a single stranded molecule

southern blotting process

• Denature DNA in alkaline solution -> separates double stranded DNA into single stranded DNA

• Transfer overnight using buffer solution -> buffer carries DNA from gel to the nylon membrane

• DNA adheres to membrane whilst the buffer is soaked up by the paper towels

northern blotting process

• No need to denature RNA -> already single stranded

• Transfer overnight using buffer solution -> buffer carries RNA from gel to the nylon membrane

• RNA adheres to the membrane whilst the buffer is soaked up by the paper towels

hybridising a labelled probe to the DNA or RNA of interest after blotting (explain)

• Remove nylon membrane with adhered DNA or RNA

• Labelled probe added in a buffer to the membrane -> leave overnight 

• Labelled probe hybridised to complementary DNA (southern) or RNA (northern)

• Probes that did not hybridise rinsed off

• Allows for detection of DNA or RNA that hybridised with labelled probes

synthesis of labelled DNA probe

• Double stranded DNA denatured to single stranded DNA templates

• Add DNA primers -> DNA polymerase synthesise new strand with labelled dNTPs

• Synthesis of labelled probe

southern blotting used for DNA analysis

• identify and quantify related genes

• determines size of related genes

• construction of restriction maps

northern blotting used for RNA analysis

• identify and quantify related RNA

• determine size of related RNA

• determines patterns of gene expression

in situ hybridisation

• Uses labelled complementary DNA or RNA probe to localise a specific DNA or RNA sequence in a portion or section of tissue or cell (in situ)

• Labelled probes inserted into cell/tissue in opposition to blotting which is inserted into gel electrophoresis

in situ hybridisation to mRNAs (antisense vs sense probe)

• Used to determine expression patterns of specific genes in tissues during development

1. Antisense probe

• Labelled probe that is complementary to the transcript

• Hybridisation occurs

2. Sense probe

• Same sequence as transcript and therefore no hybridisation

• Used as a negative control

fluorescent in situ hybridisation is used to:

detect specific DNA sequences on isolated chromosomes

ddNTPs stand for:

dideoxyribonucleotide triphosphates

ddNTPs can cause chain (?) during DNA synthesis

termination → due to the lack of a 3’ OH on the sugar of ddNTPs

sanger sequencing in 4 reaction tubes

• 4 reaction tubes -> each tube contains all dNTP type + primer + single stranded template DNA + DNA polymerase but only one of four ddNTPs

• the ddNTP determines at which nucleotide chain termination occurs → different for each reaction tube

sanger sequencing in one tube