Genomes Flashcards
1
Q
satellite DNA
A
- short sequences of repeating DNA codes within introns, telomeres (ens of chromosomes) and centromeres
- non-coding
- closer related you are, the more similar your satellite DNA will be
2
Q
mini-satellites
A
sequences of 20-50 base pairs repeating 50-hundreds of times
3
Q
micro-satellites
A
sequences of 2-4 base pairs repeated 5-15 times
4
Q
comparative genome mapping
A
- allows examining of evolutionary relationships
- identify essential genes for life
- compare pathogenic and non-pathogenic individuals
- identifying genetic polymorphisms - different alleles for a gene
- DNA profiling
5
Q
DNA profiling
A
- extract DNA - small amount from sample then replicate using PCR
- DNA strands cut into fragments by restriction enzymes at specific sites
- electrophoresis - separates DNA fragments
- hybridisation - fluorescent DNA probes added in excess to DNA fragment that bind to a known complementary sequence
- if radioactive labels added - x-ray images taken, if fluorescent labels added - placed under UV light
6
Q
uses of DNA profiling
A
- forensic science - crime
- paternity tests
- identifying who is at risk of developing a disease
7
Q
polymerase chain reaction (PCR)
A
- makes millions of copies of a fragment of DNA a few hundred bases long
1. DNA is extracted and heated to 95 degrees to break H bonds between strands - denatured
2. mixture cooled to 50-65 degrees and a primer is added which anneals (binds) to the DNA ends - needed for replication
3. mixture heated to 72 degrees and DNA polymerase added to line up free nucleotides along each template strand
4. 2 copies of DNA are formed
8
Q
what is different about the DNA polymerase used in PCR?
A
it’s derived from a thermophilic bacteria (Taq) so it doesn’t denature during extreme heating
9
Q
restriction enzymes
A
- cut DNA to get a DNA fragment from a genome
- they recognize specific palindromic sequences (same forwards and backwards eg. GGATCC)
- DNA sample incubated with specific restriction enzyme which cuts out a specific fragment by a hydrolysis reaction
- unpaired bases at the end of the fragment are called sticky ends - can anneal (bind) the DNA fragment to another piece of DNA with complementary bases
10
Q
electrophoresis
A
- DNA is digested by some restriction enzymes
- a fluorescent tag if added to the DNA fragments so they can be viewed under UV light
- DNA fragments are put into wells in agarose gel strips containing a buffer to maintain the pH
- DNA fragments of a known length are used as a reference for the fragment size - electric current is passed through the electrophoresis plate and DNA fragments in the wells at the cathode end move through the gel to the anode side because of the negatively charged phosphate groups in DNA fragments
- smaller fragments move faster as they can fit through the gel mesh
- gel is immersed in alkali to denature the DNA - separate to 2 strands and expose bases
- single stranded DNA fragments transferred to nylon membrane which is placed over the gel
- membrane covered in absorbent paper to draw the alkaline solution containing DNA through the membrane
- DNA probes are then used to allow DNA bands to be visible
11
Q
DNA probes
A
- used to identify specific DNA fragment sequences - used to locate a gene on a chromosome
- short strands of DNA with complementary bases to sequence on the gel
- they bind to the sequence you want to find
- can be attached to radioactive/fluorescent labels to allow DNA bands to be visible
12
Q
human genome project
A
- started in 1990 to map the entire human genome and make the data accessible to all scientists
- started with sequencing bacteria before applying the technique to humans
13
Q
Sanger genetic sequencing
A
- similar process to PCR is performed on the DNA - mixed with DNA polymerase, a primer, normal nucleotides and terminator bases
- Each time a terminator base is included instead of a normal nucleotide, the synthesis of DNA is terminated
- eg. an A terminator will stop DNA synthesis where an A base would be added - this results in many DNA fragments of different lengths
- After many cycles, all possible DNA chains are produced and they are separated according to length by capillary sequencing (similar to electrophoresis)
- the fluorescent markers the terminator bases can show the final base of each fragment - lasers detect the colours and the order of the sequence
- The sequence detected by the lasers is the complementary sequence to the original DNA - fed into computer that reassembles genome by comparing fragements
14
Q
differences between original and next generation Sanger sequencing
A
- original - used radioactive or fluorescent labelling of ddNTP’s , modern - uses fluorescent labelling
- original - used gel electrophoresis to separate DNA fragments, modern - uses capillary eectrophoresis
- original - involved manual reading of the DNA bands, modern - lasers detect colours and order of DNA bands
- original - slower, less cost-effective, modern - faster, more efficient
15
Q
next generation sequencing
A
- millions of fragments of DNA put on a flow cell (plastic slide) and replicated using PCR to form clusters of identical DNA fragments
- uses same principle of adding coloured terminator base so an image can be taken
- all clusters are being sequenced at the same time
- uses very high-tech computers - human genome could be sequenced in days - much cheaper
16
Q
bioinformatics
A
- development of software to analyse enormous quantities of data being generated
17
Q
computational biology
A
- uses data being analysed by bioinformatics to build models of biological systems to predict circumstances
- uses computational techniques to analyse huge amounts of biodata
- eg. helps use info from DNA sequencing to identify genes linked to diseases
18
Q
what does analysing genomes of pathogens enable?
A
- findings source of infection eg. bird flu
- identify antibiotic-resistant strains of bacteria so antibiotics are only used when needed
- tracking progress of an outbreak
- identify regions in pathogen genome that may be useful in development of drugs
19
Q
proteomics
A
- study of amino acid sequence of an organism’s whole protein complement
20
Q
spliceosomes
A
- enzymes that join exons to be translated together to give mature mRNA
- may join the same exons in a variety of diff ways - single gene produces diff versions of mRNA - codes for diff amino acids so diff phenotypes
21
Q
synthetic biology
A
- includes genetic engineering, using enzymes in drug production, synthesis of new genes to replace faulty ones, synthesis of entirely new genomes
22
Q
recombinant meaning
A
- combining DNA from more than one source into a single organism
23
Q
genetic engineering - 1. isolating the desired gene
A
technique 1. restriction enzymes cut DNA from organism leaving sticky ends
technique 2. isolate mRNA and using reverse transcriptase enzyme to produce strand of complementary DNA - easier to identify desired gene as a particular cell makes a specific type of mRNA
24
Q
genetic engineering - 2. placing the gene in a vector
A
- plasmids are used as they replicate independently - combine with host’s DNA to form recombinant DNA
- plasmid is chosen because they have a marker gene enabling scientists to dtermine the plasmid has been taken up - eg. antibiotic resistance
1. plasmid is cut using same restriction enzyme - complementary to sticky ends of plasmid
2. once the DNA fragment is lined up with the plasmid, DNA ligase forms phosphodiester bonds between sugar and phosphate groups on the strands - they usually are given a second marker gene to show that it contains the recombinant gene
- if DNA is placed in correctly, the marker gene won’t function eg. antibiotic resistance or fluorescence
25
Q
genetic engineering 3. transferring the vector
A
transformation:
- method 1 - culture bacterial cells and plasmids in solution with Ca2+ and increase temp - bacterial membrane becomes permeable and plasmids enter
- method 2 - electroporation - small electrical current applied to bacteria - makes membrane porous and plasmids move into cells
26
Q
A
