Manipulating genomes Flashcards
(23 cards)
What is genetic fingerprinting?
Technique used to identify unique DNA patterns in individuals helping identify individuals in forensics or family relationships
What does genetic fingerprinting rely on?
Every person’s DNA sequence being distinct due to variation in sequence and length of unique non-coding repetitive DNA segments called variable number tandem repeats
What are key features of variable number tandem repeats?
- present across genomes of most eukaryotes
- not involved in protein coding
- have extensive variability in sequence and length among individuals
- length and location are heritable
- chance of 2 individuals who aren’t identical twins sharing identical VNTR patterns is low
- high similarity in VNTR patterns indicates 2 individuals may be closely related
What are short tandem repeats?
Repeated sequences of nucleotides that are smaller than VNTRs and can also be used in genetic fingerprinting
Outline the process of creating a genetic fingerprint?
1) DNA is extracted from tissue sample and amplified using PCR
2) restriction enzymes used to cut DNA into fragments at points near VNTR sequences
3) gel electrophoresis separates fragments by size and they’re denatured producing single strands
4) specific radioactive or fluorescent probes bind to complementary VNTR sequences
5) positions of probes revealed creating barcode-like pattern of DNA bands unique to individual
What happens after radioactive probes are used in genetic fingerprinting?
X-ray images taken of paper or membrane during development stage
What happens after fluorescent probes are used in genetic fingerprinting?
Paper or membrane placed under UV light causing fluorescent probes to glow
How can probability of a match be determined in genetic fingerprinting?
Computer analysis of band patterns
higher number of matching bands greater likelihood fingerprints are from same individual
What are key uses of genetic fingerprinting?
- establish paternity by matching bands on child’s genetic fingerprint to potential parents and assessing similarities between VNTR patterns
- identifying suspects from crime scene DNA (e.g blood)
- support criminal convictions with match probability calculations
- identify risk of genetic disorders and predicting onset and severity
- select desirable traits in plants and animals for selective breeding while preventing severe inbreeding
- evaluate genetic diversity by comparing variety of genetic fingerprints within a population
What are limitations of genetic fingerprinting?
- environmental contamination may compromise results
- close genetic relatives could have similar fingerprints
- assumptions about genetic variation in populations underpin probability calculations meaning doesn’t prove guilt or causation
What is polymerase chain reaction (PCR)?
Method for amplifying DNA fragments rapidly and efficiently
What are the components required for PCR?
DNA fragment = specific target DNA template sequence needing replication
Primers = short sequences of nucleotides attaching to start and end of DNA fragment to be copied
DNA polymerase = enzyme creating new DNA strands by adding DNA nucleotides
Free nucleotides = building blocks (A, T, C, and G) used to construct new DNA strands
Thermocycler = device that precisely heats and cools PCR mixture to facilitate reaction
What type of DNA polymerase is used in PCR and give an example?
One that can withstand high temperatures without denaturing ensuring it remains active throughout process
e.g Taq polymerase from thermophilic bacteria
What happens during the 1st stage of PCR and at what temp?
Separation (denaturation) = heating DNA separates H bonds between complementary strands
95°C
What happens during the 2nd stage of PCR and at what temp?
Primers added (annealing) = primers attach to specific starting points on each of separated DNA strands by forming H bonds
55°C
What happens during the 3rd stage of PCR and at what temp?
DNA synthesis (extension) = DNA polymerase adds free nucleotides to ends of primers extending DNA strand to form a complete copy
72°C
What are advantages of PCR?
Rapid speed = produces approx 100 billion copies in few hours
Precision = automated nature of thermocycling ensures accurate amplification of DNA fragment
Low DNA needs = tiny samples amplified producing large quantity for analysis
No cells needed = simpler and more straightforward than traditional in vivo techniques
What is gel electrophoresis?
Technique separating molecules such as DNA, RNA, or proteins based on size using electric current applied to an agarose gel matrix
How do you set up gel electrophoresis?
1) insert gel tray with solidified agarose gel into gel tank
2) ensure wells are close to negative electrode to position gel correctly
3) pour buffer solution over gel until submerged to maintain constant suitable pH throughout experiment
How do you load samples in gel electrophoresis?
1) mix DNA or RNA samples with loading dye to make visible
2) carefully deposit equal volumes of samples into wells using micropipette
3) touch micropipette tip to buffer not bottom of gel preventing gel damage
4) keep record of which sample is in each well for later analysis
What charge do RNA and DNA molecules carry and why?
negative charge
due to their phosphate groups
What happens during gel electrophoresis?
1) voltage (around 100 V) applied across gel
2) fragments of DNA or RNA move towards positive electrode (anode)
3) smaller fragments travel faster and thus separate by size
4) continue process for about 30 minutes or until dye approaches end of gel
How do you visualise results of gel electrophoresis?
1) switch off voltage and remove gel from tank
2) apply stain to DNA or RNA to reveal bands of fragments
3) assess migration distances of bands to approximate sizes of fragments
