Module 8 quick Flashcards
DNA fragments reverse transcription
Makes dna copies from mRNA
Occurs naturall in viruses
Cell that naturally produces the protein of interest is selected
Cells have lots of mRNA for that protein
Reverse transcriptase joins dna nucleotides complementary bases to the mRNA sequence
Single stranded in torn free cDNA makes double stranded using DNA polymerase
Use PCR
Restriction endonucleases
Bacteria use as a defense
Complementary active site to the recognition sequence
Some cut at the same location to make blunt ends
Some cut palindromic to make sticky ends which join dna with a complementary base pair
Gene machine
Uses a machine
Examine the protein to identify the amino acid sequence
Work out mRNA and DNA sequence
Enter the sequence to the computer, checks biosafety and biosecurity
Creates small overlapping single strands called oligonucleotides
Joined to make entire gene dna
PCR can amplify quantity
In Vivo cloning
Restriction endonucleases cut gene at sticky ends
Modify by adding a promoter region to the start as a rna polymerase binding site and a terminator region to the end and rna polymerase detachment site
Restriction endonucleases are used to cut open the plasmids, same as the one used to cut the gene so the same sequence is cut so the sticky ends are complementary
DNA ligase annuals the dna fragments, catalysts the formation of phosphodiester bonds between bases
Vector placed in host cell, made more permeable by adding calcium ions and. Heat shock
So the vector can enter the cytoplasm
Not all the bacteria will take up the plasmid because the plasmid may not be inside, plasmid rejoins before the fragment enters,, fragment sticks
In vivo cloning with antibiotic resistance
Resistance to tetracycline and ampicillin
Insert fragment into tetracycline so plasmid won’t be resistant to tetracycline
Grow on agar
Use master plate stamp onto a sterile block and stamp agar with ampicillin
Stamp onto a new agar plate with tetracycline on the agar
Colonies on the plate don’t have the gene
Compare to the previous plate to see which grew
Grow these in a fermenter
In vivo cloning with fluorescence
GFP from jellyfish GFP inserted into a bacterial plasmid Insert fragment to the middle of GFP Grow agar and expose to us light If the GFP glows then no gene If the GFP doesn’t glow then the plasmid has combined Grow these ones in a fermenter
In vivo cloning using genes coding for enzymes
Lactase goes blue to colourless Insert into the plasmid Fragment in the middle Grow and turn colourless If it cant turn blue then contains the plasmid
DNA probes
Short single stranded sections of dna that are radioactive/fluorescently labelled
Locate specific alleles
Creates probe that is complementary to allele
Patients dna that is single stranded is mixed with probes
Patient has allele dna probe will hybridise (heat to break hydrogen bonds, mixed with probe and cooled, anneal and reform hydrogen bonds), label indicates presence
Locating specific alleles
Use a known DNA sequence Probe Use dna sequencing techniques Fragment made in the gene machine Amplify in the PCR Add a radioactive or fluorescent label Wash If fluorescent then prescence
Screening
Use an array or microarray
Glass slide with probes on
Add sample
Genetic fingerprinting
95% of dna are introns which are VNTRs, variable number tandem repeats
Collection/extraction:
-smallest sample of blood or cells or hair, if small then use PCR to amplify
Digestion:
-restriction endonucleases cut dna into fragments
-enzymes which cut close to target VNTRs are added
Separation:
- dna to wells in agar, gel in buffer liquid with electrical voltage applied
-dna has a negative charge so dna move through gel to positive end
-gel electrophoresis
-agar creates a resistance for dna, smaller pieces move faster and further
-VNTRs separate
-alkaline is added to separate double dna strands
Hybridisation
-dna probes are short, single stranded dna pieces complementary to VNTRs, probes radioactively/fluorescently labelled
-different probes mix with single stranded dna VNTRs on agar gel they hybridise Rinse to remove non-blinded
Development:
-VNTRs and probes onto nylon sheet, agar will crack and shrink
-expose to x rays or uv light
Analysis:
-position of bands are compared to identify relationships
-use marker with known VNTRs to compare to
In vitro cloning
Once fragments are sorted need to be cloned
Fragments can be amplified via PCR
Need a thermocycler, DNA Taq polymerase, primers and nucleotides
Heat 95 degrees, break hydrogen bonds, make dna single stranded
Temp 55 degrees so primers can anneal
Taq DNA polymerase attaches to complementary free nucleotides , new strand to align ext to each template synthesis increase to 72 degrees which is optimum for Taq polymerase
