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
Why is it better to start with mRNA from pancreas cells rather then using their DNA
-Doesn’t have to be spliced to remove introns
-Specific mRNA is found in pancreas cells
-There are multiple copies of mRNA
Which enzyme converts mRNA into single-stranded DNA
Reverse transcriptase
Which enzyme converts single-stranded DNA into double-stranded DNA
DNA polymerase
What do DNA markers consists of
Fragments of DNA of known size
In which stage of the cell cycle do spindle fibres form
Metaphase
In which stage of the cell cycle does DNA replication occur
Interphase
In which stage of the cell cycle do sister chromatids get pulled to opposite poles of the cell
Anaphase
In which stages of the cell cycle are chromosomes not visible
Interphase and Telophase
In which stage of the cell cycle does the nuclear membrane break down
Prophase
In which stage of the cell cycle does the nuclear membrane reform
Telophase
In which stages of the cell cycle is the DNA presented as chromatins
Interphase and Telophase
In which stage of the cell cycle are 2 daughter cells produced
Telophase
In which stage of the cell cycle do the chromosomes begin to shorten and thicken
Prophase
In which stage of the cell cycle do the chromosomes move to the equator of the cell
Metaphase
In which stage of the cell cycle are cell organelles replicated
Interphase
In which stage of the cell cycle is DNA polymerase activated
Interphase
In which stage of the cell cycle do the centrioles move to the poles
Prophase
