C21 - Manipulating genomes Flashcards
What is a genome?
All genetic material of an organism
What are exons?
DNA that codes for proteins (2% of total DNA)
What are introns?
Regions of non coding DNA/RNA
What is satellite DNA?
Short sequences of DNA repeated many times
eg. introns, telomeres, centromeres
What are the two types of satellite DNA? How do you differentiate?
Minisatellite - sequence of 20-50 base pairs repeated 50-100s of times
AKA variable number tandem repeats VNTRs
Microsatellite - 2-4 base pairs repeated 5-15 times
AKA short tandem repeats STRs
What aspects of the satellite DNA stay the same/change between people’s DNA?
Position of mini/micro-satellites remain same on chromosome
Number of repeats varies between individuals
What do similarities in satellite patterns tell us?
Typically have a more similar satellite pattern to someone we are closely related to (inherited)
What is DNA profiling?
Produces image of patterns in non-coding DNA of an individual
How do you produce a DNA profile?
1) Extract DNA
Using polymerase chain reaction (PCR)
2) Digest the sample
Strands of DNA cut into small fragments using enzymes called restriction endonucleases
(dif RE cut at dif restriction site)
3) Separate the DNA fragments
Use electrophoresis
Charged particles move through gel medium under influence of current, gel immersed in alkali to separate DNA into single strands
DNA fragments transferred to nylon membrane using Southern blotting
4) Hybridisation
Radioactive/fluorescent DNA probes (short sections of complimentary DNA/RNA) added to DNA fragments
DNA probes bind to DNA under specific pH and temp
Probes identify varied regions
Excess probes washed off
5) See the evidence
For radioactive labels, X-Rays used
For fluorescent, membrane placed under UV light
What is polymerase chain reaction?
Process by which small sample of DNA can be amplified using specific enzymes and temp changes
What are restriction endonucleases?
Enzymes that chop strand of DNA into small pieces
What is electophoresis?
Type of chromatography that relies on the way charged particles move through a gel under the influence of an electric current, used to separate nucleic acid fragments or proteins
How does PCR work?
1) Seperate stands
-Temp in PCR is inc to 90-95C for 30secs
-Denatures DNA by break H bonds
-DNA strands separate
2) Annealing of primers
-Temp decreased to 55-60C
-Primers bind (anneal) to ends of DNA stands
3) Synthesis of DNA
-Temp increased to 73-75C, for min 1min
-Optimum temp for DNA polymerase
-DNA polymerase adds bases to primer, building complimentary strands of DNA
-Enzyme Taq polymerase used
What is DNA profiling used for?
-Forensics
-Paternity tests
-Identifying species + evolutionary relationships
-Identifying risk of developing certain diseases
How did DNA sequencing begin?
Radioactive labelling of bases and gel electrophoresis
Initially manual until Sanger sequencing invented which read multiple bases at a time
What was the human genome project?
Scientists in many countries worked to map the entire human genome
How is DNA sequencing carried out?
1) DNA mixed with primer, DNA polymerase, excess of normal nucleotides, terminator bases
2) Placed in thermal cycler that rapidly changes temp
3) 96C - double stranded DNA separates to single strands
4) 50C - primers anneal to DNA strands
5) 60C - DNA polymerase builds new DNA strand, adding nucleotides w complimentary bases to template DNA strand
6) Synthesis of DNA stops when terminator base added, many dif length DNA fragments
7) DNA fragments separated based on length by capillary sequencing - similar to gel electrophoresis in small capillary tubes
8) Dif coloured Fluorescent tags on terminator bases help identify base + order of sequence
9) Order of bases in capillary tubes show complimentary strands of DNA, info used to build of DNA
What is next generation sequencing?
Massively parallel sequencing
FasterSequencing
reaction takes place on flow cell (plastic slide) rather than gel/capillaries
Fragments do DNA replicated using PCR,
What is computational biology?
Study of biology using computational techniques to analyse large amounts of data
What is bioinformatics?
Development of software and computing tools needed to analyse and organise raw biology
What does sequencing the genomes of pathogens enable?
Dr can find source of infection
-Identify antibiotic strains of bacteria (enable correct prescription of antibiotics)
-Scientists track progress of outbreak of diseases
-Identify regions in genome of pathogens that may be useful targets for drug development/genetic markers for vaccines
What is DNA barcoding?
Technique to identify particular sections of genome that are common to all species but vary between them
What is the international barcode of life project?
Scientists identify species using short sections of DNA from conserved region of genome
What does DNA barcoding not work for?
Fungi, bacteria
How can genome sequencing be used to understand evolutionary relationships?
Basic mutation rate of DNA calculated
Can calculate how long ago two species diverged from common ancestor
