Topic 15.1: Nucleic acid hybridisation

Question 1

protein level - structural level

Answer 1

gel electrophoresis

Question 2

protein level - functional level

Answer 2

enzyme assays

Question 3

most molecular techniques investigate at

Answer 3

DNA level

Question 4

DNA level - nucleotide level

Answer 4

DNA sequencing
PCR plus sequencing
PCR plus restriction analysis

Question 5

DNA level - gene level

Answer 5

DNA sequencing
PCR technology
southern hybridisation
microarrays
DNA profiling

Question 6

DNA level - chromosome level

Answer 6

karyotyping
fluorescent in situ hypbridisation (FISH)
chromosomal painting

Question 7

DNA level - genome level

Answer 7

genome sequencing and analysis
whole chromosome sequencing
microarrays
exome sequencing
microbiomes

Question 8

what is nucleic acid hybridisation

Answer 8

DNA molecule’s ability to denature (double to 2 single stranded) and renature (double stranded)
introduce a complementary sequence to one of the strands = DNA probe

Question 9

what is southern blotting

Answer 9

combined gel electrophoresis and DNA hybridisation:

1. separate fragments
2. detect particular sequence with specifically labelled probes that will bind

Question 10

how does DNA hybridisation - southern blotting work

Answer 10

1. GEL ELECTROPHORESIS on agarose gel = fragments
2. transfer fragments onto nitrocellulose gel
3. add labelled DNA probe (usually single stranded) - flourescent
4. probe binds to complementary sequences
5. detect using autoradiography or flourescence imaging (probe binds but not light up..its the actual DNA its binding to only which is detected)

Question 11

what are the characteristsics of probe in southern blotting

Answer 11

enough similarity to bind - the odd base is not complementary
not have to completely align with target sequence

Question 12

what are the uses of southern hybridisation

Answer 12

analyse clones
investigate gene structure - any deletions or duplication
investigate gene expansions - triplet repeats for Huntigton’s
investigate mutations using allele specific probes - sickle cell
dna fingerprinting

Question 13

what are microarrays

Answer 13

multiple genes expressed in a single place attahced to a solid support…can make single stranded oligonucleotides which are specific to a sequence of a particular gene. binds them on lots of genes due to sequence being complementary to coding region of genome

Question 14

what do microarrays do

Answer 14

monitor changes in gene expression - see if mRNA binds, eg: normal cell -> tumour cells

Question 15

how do microarrays work

Answer 15

1. isolate mRNA
2. convert to cDNA (which is complementary to target gene) using reverse transcriptase and incorporate colored dNTPS (separate colours for normal cells and tumour cells)
3. add to microarray slide
4. flourscent strands anneal to complementary sequences
5. wash away unhybridisated cDNA strands and analyse…if more of colour…more of the mRNA of that cell being expressed..which genes expressed

Question 16

what is FISH

Answer 16

identify DNA sequences within cells or chromosomes
probes against particular DNA sequences, incorporated into isolated cells and see if flourescentally labelled probes bind to target sequence on chromosome

Question 17

what can FISH be used for

Answer 17

chromosome 21 x3
chromosome 13 x2
= down syndrome (triosomy 21)