Gene Cloning 2

Question 0

how does in situ hybridisation work ?

Answer 0

an antisense RNA probe is produced to hybridise to the specific mRNA that you want to detect
it enables the quantification of the levels of mRNA expression in cells
it can be amplified by labelling the probe with enzymes

Question 1

how can we tell which cells express a gene ?

Answer 1

in situ hybridisation

Question 2

what did in situ hybridisation show about the kainate receptors in a control brain and the brain of a schizophrenic brain ?

Answer 2

the level of GluR6 subunits were lower in schizophrenic brain however it was difficult to quantify, but it provided spatial and temporal information

Question 3

what is reverse transcriptase- polymerase chain reaction (RT-PCR)?

Answer 3

a way of measuring gene expression
can analyse and amplify small amounts of DNA
- an oligoDT primer/random primer/gene specific primer can be used to bind to the specific gene
- this pulls out the mRNA strand which can then be reverse transcribed by reverse transcriptase to produce cDNA
- the cDNA is used as a template in PCR

Question 4

how does the PCR process work ?

Answer 4

• the gene specific primer is hybridised to a single strand length of DNA
• this primer is then extended using Tac Polymerase and dNTPs (Tac works at high temps and in 5’ to 3’ direction)
• this creates a double stranded length of DNA
• this is then heated to about 95 degrees to break hydrogen bonds to produce to single strands
• temperature is cooled to about 50-60 degrees to anneal primers to each of the strands - more primers are added compared to strands to ensure the primers bind to their specific strand
• heated to 72 degrees which is optimal temp for TAC so primers add nucleic acids to produce 2 double stranded lengths of DNA

Question 5

what are the advantages of PCR?

Answer 5

• only requires 1 cell
• most sensitive technique
• most useful thing is it can clone genes and introduce changes in the DNA sequence

Question 6

what are the disadvantages of PCR?

Answer 6

• cant look at all different spliced variants at once, the appropriate design of primer sequences has to be produced to analyse different spliced variants
• it can be difficult to quantify mRNA because of its sensitivity - any errors are greatly amplified and the reaction will saturate eventually
• size of mRNA cannot be determined

Question 7

what does the northern blot require ?

Answer 7

requires large amount of mRNA

Question 8

how can the coding region for a specific gene be amplified ?

Answer 8

• firstly cDNA is produced
• using gene specific primer to amplify an mRNA of interest- linker sequence with restriction enzymes is added so the DNA can be digested and inserted into a plasmid- a sense and antisense primer are added
• then the PCR process is carried out
• this produces the cDNA of the specific coding region which can be cloned into a plasmid

Question 9

what is alternate splicing ?

Answer 9

mRNA is made up of many exons and introns
the introns are spliced out as they are non- coding
the annealing together of different exons can be joined together differently enabling alternate splicing

Question 10

what are the 2 different exons in the GluR1 gene ?

Answer 10

mRNA of GluR1 contains either the flop or flip exon but never boh

Question 11

what was shown in the exon experiment of the GluR1 gene ?

Answer 11

when GluR1 has flip exon and GluR2 has flop exon there is a high transient current but the sustained current is lost
when GluR1 has flop exon and GluR2 has flip exon the transient current is lost but the sustained current is current

Question 12

what happens during development for the exons present in the GluR receptors ?

Answer 12

during development the flop variants are expressed at only low levels
the inclusion of the flop exon increases with maturation

Question 13

why cant proteins be identified by hybridisation ?

Answer 13

because nucleic acids have complementarity whereas proteins dont therefore they cant be identified by this method

Question 14

what are produced to identify proteins ?

Answer 14

specific antibodies

by using immune systems

Question 15

how can you get a protein to inject into an animal ?

Answer 15

• get a protein from somewhere such as a liver and then homogenise it and purify it and then this can be injected into an animal
• can clone a protein by cloning a gene to produce cDNA which can be inserted into a plasmid and then put into a bacteria and then it can be purified and injected into an animal
• chemically synthesise a short peptide by adding a single amino acid one at a time to a glass tube

Question 16

how are antibodies produced for a protein ?

Answer 16

take an animal and inject a protein into it
then the animal will produce antibodies against this
then extract some serum from the animal and this will contain the antibody for that protein and this can then be used as a probe

Question 17

how are western blots carried out ?

Answer 17

sample is electrophoresed through a polyacrylamide gel to separate by size
this is transferred to a nitrocellulose membrane
this is incubated with antibody
identify antibody by an enzyme linked secondary antibody and a light producing reaction

Question 18

what can a western blot tell you ?

Answer 18

tell you the size and relative abundance of proteins, identify splice variants or posttranslational modifications

Question 19

what does it mean if the western blot produced has 2 different markers?

Answer 19

antibody has identified 2 signals
-could be alternative forms of the protein - different exons being used or the protein has be poststranslational modified

gives a rough idea of their size

Question 20

what is immunocytochemistry and immunohistochemistry ?

Answer 20

similar to in situ hybridisation

incubate with antibodies are the probe and this tells you which cells this protein is expressed in