Semester 1 Biology part 2 Flashcards
Restriction endonucleases
-Cuts desired gene (from DNA) of desired organism
-Recognition sites are palindromic
DNA ligase
-Can rejoin sticky ends between nucleotides, reforms the sugar phosphate backbone
-Can return plasmid to bacterial cells
Process of genetic engineering
-Genes are taken from one organism and inserted into another host organism
-Alters genetic makeup of organism=transgenic organism
-Bacteria, yeast, viruses (phages) usually used as recipient cells
-The rapid reproduction of these microorganisms enables transferred gene to be amplified
In vivo
Where copies are made inside a living organism
In vitro
Where copies are made outside a living organsim e.g a lab/test tube
Outline the steps of PCR
-Reaction mix contains primers, template DNA, nucleotides and DNA polymerase
-Mixture heater to 95 deg, H bonds are broken between bases, strands separate
-Cool to 45-65 deg so primers bind to DNA strands
-Increase temp to 70-75 deg, DNA polymerase joins adjacent nucleotides together via complementary base pairing
-2 double stranded molecules are produced
-Repeat cycle many times
What factors limit PCR
-Primers running out
-Nucleotides run out
-Enzymes denature
Why are 2 different primers used in PCRs
-Sequences at the end of target sequence are different
-One is at the beginning and one is at the end
Formula for calc DNA strands
2^n
but remember to X2 if asking for the number of strands as DNA is double stranded
What is a primer
-A short single stranded DNA
-Its bases are complementary to the part of the DNA to be copied
Why are primers used
-Defines the section to be copied
-Allows attachment of polymerase
Define Genetic markers
-They enable genetically engineered bacteria to be detected and isolated for culturing
Antibiotic resistance marker genes
-Used to be used as marker genes,
-But due to risk of spread of antibiotic resistance by horizontal gene transmission between species safer methods have been developed
Flurorescent marker gene
-GFP gene codes for production of green fluorescent protein
-Cloned gene is added to GFP gene
-Successfully transformed bacteria are identified under UV light as they fluoresce
How to obtain gene products (plasmid)
-Bacteria that takes up the recombinant plasmid will replicate during cell division producing a colony
-Bacterial colonies can be harvested and stored
-Plasmid can be purified, digested with restriction endonucleases and analysed using gel electrophoresis to confirm presence of foreign DNA
-Bacterial cells are cultured and produce desired gene product in large amounts.
How does reverse transcriptase work in producing a required gene
-mRNA is used as a template to produce required gene
e.g to produce insulin mRNA strand complementary to insulin gene is used
-mRNA mixed with free DNA nucleotides and reverse transcriptase enzyme
-Reverse transcriptase joins adjacent nucleotides together to produce fragment of DNA
-DNA produced is called cDNA
-cDNA can be converted into double stranded DNA using DNA polymerase
Describe how electrophoresis works
-DNA sample is extracted, mixed with a loading buffer
-Placed into wells and a current is applied
-DNA migrates towards the anode (positive electrode) due to DNA’s negative phosphate group
-DNA separates according to size, smaller fragments travel quicker and larger fragments get trapped
-Fragments can be stained with Fluorescent dye and placed under UV light
-Gel can be photographed to record the position and fragments of DNA can be extracted from gel
Gene probes
-Short single-stranded lengths of DNA (15-20 bases long)
-Its bases are complementary with the DNA/allele/gene
What does a radioactive probe do
-Binds to specific base sequences
-Makes DNA visible using autoradiography
Describe the procedures involved in the production of a genetic fingerprint from a sample of DNA taken from a crime scene. (6 Marks)
-DNA is cut;
-using restriction enzyme;
-electrophoresis;
-separates according to length/mass/size;
-DNA made single-stranded;
-transfer to membrane/ Southern blotting;
-apply probe;
-radioactive/ single stranded/ detected on film/ fluorescent;
-reference to tandem repeats/VNTRs/minisatellites;
-patten unique to every individual:
Uses of genetic fingerprinting
-Forensic science, characterise victims using small amounts of genetic material
-Phylogenetic studies, allows evolutionary links to be investigated
-Screening of blood and blood products , to detect contamination by viruses to prevent further infections
-Pateint monitoring, Success of treatments e.g chemotherapy can be followed. DNA produced can be used in gene therapy
-Genetic screening, collecting info about different diseases especially those that can be passed onto offsprings
What is DNA sequencing used for
-Used to determine sequence of nucleotides in a sample
-DNA sample used may have been obtained from southern blotting
What will the reaction tube contain for DNA sequencing and state the role of each
-Large quantity of DNA
-DNA primers, radioactively labelled 32^P (starts the sequencing and radioactivity allows presence of primer to be detected)
-All 4 DNA nucleotides: ATCG (align via CBP)
-DNA polymerase (joins nucleotides together)
-Terminator nucleotides (terminates the extension of DNA strand as it forms dideoxy nucleotide)
Outline the process of DNA sequencing
-DNA polymerase uses radioactive primer to form complementary DNA strand
-DNA nucleotides are joined together using sample of DNA as template
-The random addition of chemically modified nucleotide stops synthesis of DNA strand… majority of reaction continues but can be terminated later on
-Due to random termination a tube will have a set of different sized DNA fragments as DNA molecules are terminated by different nucleotides at diff positions
-All fragments are radioactive due to their primers
