Gene Tech & applications Flashcards
Describe how DNA is replicated in a cell
- DNA strands separate / hydrogen bonds broken;
- Parent strand acts as a template / copied / semi-conservative replication;
- Nucleotides line up by complementary base pairing; (Adenine & Thymine etc)
- Role of DNA polymerase: joins adjacent nucleotides on the developing strand via condensation and formation of phosphodiester bond;
- 5’ to 3’ direction
- Each new DNA molecule has 1 template and 1 new strand
- Formed by semi-conservative replication.
Why is the DNA heated to 95°C during PCR?
- Produce single stranded DNA
- Breaks WEAK hydrogen bonds between strands
Why do you add primers during PCR?
- Attaches to / complementary to start of the gene / end of fragment;
- Replication of base sequence from here;
- Prevents strands annealing
Explain why ‘base-pairs’ is a suitable unit for measuring the length of a piece of DNA.
- DNA = 2 chains / joined by linking of 2 bases / A with T and G with C/ purine pairs with pyrimidine;
- Bases are a constant distance apart / nucleotides occupy constant distance/
- each base-pair is same length / sugar-phosphate is a constant distance;
A deletion mutation occurs in gene 1.
Describe how a deletion mutation alters the structure of a gene
- removal of one or more bases/nucleotide;
- frameshift /(from point of mutation) base sequence change;
Describe a plasmid.
- circular DNA;
- separate from main bacterial DNA;
- contains only a few genes;
Suggest one reason why DNA replication stops in the polymerase chain reaction
- Limited number of primers/nucleotides; /
Primers / nucleotides ‘used up’. - DNA polymerase (eventually)denatures
Suggest why the restriction enzyme has cut the human DNA in many places but has cut the plasmid DNA only once.
- enzymes only cut DNA at specific base sequence/recognition site/specific point;
- sequence of bases/recognition site/specific point (on which enzyme acts)
- occurs once in plasmid and many times in human DNA;
- (max 1 if no reference to base sequence or recognition site)
Explain what is meant by a vector.
- Carrier;
- DNA/gene; (context of foreign DNA)
- Into cell/other organism/host;
Explain how modified plasmids are made by genetic engineering and how the use of markers enable bacteria containing these plasmids to be detected.
- isolate TARGET gene/DNA from another organism/mRNA from
- cell/organism;
- using restriction endonuclease/restriction enzyme/reverse transcriptase to
- get DNA;
- produce sticky ends;
- use DNA ligase to join TARGET gene to plasmid;
- also include marker gene;
- example of marker e.g. antibiotic resistance;
- add plasmid to bacteria to grow (colonies);
- (replica) plate onto medium where the marker gene is expressed;
- bacteria/colonies not killed have antibiotic resistance gene and (probably) the TARGET gene;
- bacteria/colonies expressing the marker gene have the TARGET gene as well;
mRNA may be described as a polymer. Explain why.
- Made up of many (similar) molecules/monomers/nucleotides/units
describe how genetic fingerprinting is carried out
- DNA extracted from a sample
- DNA cut /hydrolysed into segments using restriction endonuclease
- Must leave VNTRs /mini satellites intact
- DNA fragments separated by electrophoresis
- Detail of process e.g mixture put into wells on gel and electric current passed through
- Immerse gel in alkaline solution / two strands of DNA separated
- Radiocative marker
- Identified using x ray film
What is recombinant DNA
- cell having 2 or more sources of DNA
what is the function of reverse transcriptase
- carries out transcription in reverse, makes cDNA from mRNA templates
what is the function of restrictive endonuclease
- hydrolyse DNA at specific recognition (base) sequences
advantage of isolating DNA fragments via gene machine
- artificial genes easily transcribed and translated by prokaryotes, as they have no introns in their DNA
what is the function of DNA ligase
join DNA fragment and vector DNA at the sugar phosphate backbone, called ligation forms phosphodiester bonds
What is a DNA probe
short, single stranded DNA molecule with complimentary base sequence to DNA fragment to be located which is radioactive
name the 5 steps in recombinant DNA technology
- isolation
- insertion
- transformation
- identification
- growth
what are 3 ways of producing fragments of DNA
- mRNA->cDNA via reverse transcriptase
- restrictive enzymes to cut fragment w desired gene from DNA
- creating gene w gene machine
outline a method for in vivo gene cloning
- cut desired gene from desired organism
- using restrictive endonuclease
- make artificial DNA w correct base sequences
- cut plasmid open
- w same restriction endonuclease
- ref. sticky ends/unpaired bases attached
- use DNA ligase to join
- return plasmid to bacterial cells
Describe the process of PCR (6 marks)
- heat DNA to 95•C, breaks Hydrogen bonds
- add primers and nucleotides
- cool to 50•C, allows binding of nucleotides
- add DNA (taq) polymerase
- heat to 75•C
- DNA polymerase joins nucleotides together
- repeat cycle many times
