paper 3 Flashcards
Reverse transcription
1) Makes DNA from mRNA
2) A cell that naturally produces a protein of interest is selected.
3) Must have a large amount of mRNA of that protein
4) Reverse transcriptase is added
5) makes a single DNA strand cDNA
6) DNA polymerase used to join 2 DNA strands togethrr
Advantage of reverse transcriptase
Intron free
Restriction endonuclease
1) enzymes that cut DNA
2) naturally occur in bacteria as a defence mechanism
3) Active site complementary shape to a range of DNA base sequences.
4) some cut at the same place causing blunt ends
5) some cut and create staggered ends exposing bases (called sticky ends)
6) Palindromic becuase they can join to DNA with complementary bases.
Gene machine
1) created in a lab using computerised machine
2) Protein of interest is examined to identify the amino acid sequence and then use that to work out what the mRNA and DNA sequence would be.
3) DNA sequence is entered into computer, which checks for biosafety and biosecurity that the DNA is safeand ethical to produce.
4) creates small sections of overlapping single strands of nucleotides that make up a gene called oligonucleotides.
5) ON can be joined to create DNA for the entire gene.
PCR can be used to amplify the quanitity and to make double strand.
Advantage of gene machine
quick
accurate
makes intron free DNA so can be transcribed into prokaryotic cells
Two ways DNA fragmets are amplified
1) in vivo cloning
2) in vitro cloning
Promoter and terminator region
Restriction endonuclease cuts at recognition site leaving sticky ends.
1) promoter region added- added at start of DNA fragment. A sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur.
2) Terminator region- added to end og gene. Causes RNA polymerase to detach and stop transcription, so only one gene is copied at a time into mRNA.
What is a vector?
Something to carry the DNA into the host cell.
commanly plasmids- seperate from main bacterial genome which only contains a few genes
Insert DNA into vector
1) plasmid cut open using same R endonuclease.
2) Creates the same sticky ends
3) DNA se complementary to plasmid se and so ligase is used to stick them together via a condensation reaction to form phosphodiester bonds between nucleotides.
How its inserted into host cell (in vivo)
1) host cells are mixed with Ca2+ and get heat shocked to make it more permeable to the vector (by causing a sudden increase then decrease in temp)
2) Enables the vector to enter the host cells cytoplasm
Issues that can occur with inserting the recombinant plasmid
1) Doesn’t get inside the cell
2) Plasmid rejoins before the DNA fragment entered.
3) DNA fragment sticks to itself rather than inserting into the plasmid
What are marker gene?
name them
1) antibiotic resistance genes
2) Genes coding for fluorescent proteins
3) Genes coding for enzymes
PCR
1) temp increased to 95, h bonds break and DNA split into single strands.
2) temp decreased to 55 so that primers can attach
3) DNA polymerase then attaches complementary free nucleotides and makes a new strand to align next to each template. Temp inscreased to 72 (oprimum for taq polymerase)
Advantage of PCR
1) automated
2) Rapid
3) Doesn’t require living cells
Explain how a single base substitution causes a change in the structure of this
polypeptide.
Do not include details of transcription and translation in your answer
- Change in (sequence of) amino acid(s)/primary structure;
- Change in hydrogen/ionic/disulfide bonds;
- Alters tertiary/30
structure;
What is a DNA probe?
- (Short) single strand of DNA;
2. Bases complementary (with DNA/allele/gene)
Describe how genetic fingerprinting is carried out. (6)
1 DNA extracted from sample;
2 DNA cut / hydrolysed into segments using restriction endonucleases;
3 must leave minisatellites / required core sequences intact;
4 DNA fragments separated using electrophoresis;
5 detail of process e.g. mixture put into wells on gel and electric
current passed through;
6 immerse gel in alkaline solution / two strands of DNA separated;
7 Southern blotting / cover with nylon / absorbent paper (to absorb DNA);
8 DNA fixed to nylon / membrane using uv light
9 radioactive marker / probe added (which is picked up by required
fragments) / complementary to minisatellites;
10 (areas with probe) identified using X-ray film / autoradiography;
What is the advantage of the enzyme used in the polymerase chain reaction being thermostable?
would not be denatured;
must be heated to 95 °C / must withstand high temps;
Why are DNA primers added during the polymerase chain reaction?
to mark beginning and / or ends of the part of DNA needed /
for attachment of enzymes or nucleotides / initiator /
keeps strands apart;
What are DNA primers?
short lengths / fragments of DNA / nucleotides /
single stranded DNA;
Explain why the DNA is heated to 95 °C.
to separate the two strands of the DNA /
to break the hydrogen bonds;
Tests for use in criminal cases often take much longer because samples are very
small or contaminated (lines 8-10). Explain why it takes longer to obtain a genetic
fingerprint if the sample is
very small;
contaminated
(i) PCR / amplification needed;
(ii) other DNA present; need to identify ‘required’ DNA from rest
Explain one way in which the polymerase chain reaction differs from DNA replication in a cell.
uses heat; to separate strands; OR PCR replicates pieces of DNA; because DNA has been cut; OR primer added in PCR; to initiate replication
What is meant by a genome?
(All) the DNA in a cell/organism;
