Manipulating Genomes Flashcards
what is the genome
entire genetic makeup
what is the proteome
all proteins produced by the genome of an organism
what do dna sequences determine
therefore dna sequencing =
the precise order of nucleotides in a DNA molecule, allowing genes to be isolated and read
= working out the sequence of bases
why does the genome have to be fragmented over time before sequencing
- genome too large
- smaller fragments make sequences more accurate (less errors)
is sanger sequencing/chain sequencing new or old
old
laborious
but more reliable
Process of Sanger Sequencing
- Add single strand of DNA template, DNA polymerase, and free (deoxy)nucleotides
(A/T/G/C) - In each tube add 1 of 4 types of dideoxynucleotides (A/T/C/G)
- If deoxynucleotide added to DNA strand, DNA replication continues
- If dideoxynucleotide added to DNA strand, DNA replication is stopped
- This produces many complementary DNA strands of different lengths
- These developing strands are separated from the template DNA
- They are separated by length using gel electrophoresis
● Gel electrophoresis has 4 wells (one for each dideoxynucleotide)
● The shorter the length of DNA, the faster it moves down the gel
● Allows the base sequence of the developing strand to be built up one base at a time
Dideoxynucleotides = modified nucleotides. how?
● Lack -OH group on 3’ carbon of sugar ring
o -OH group cannot bind to next nucleotide
o No more nucleotides can be added to chain
● When added to strand, will stop replication
● Labelled with radioactive isotope/different colour/fluorescent dye
what is Sanger sequencing
sequencing by chain termination
High-throughput sequencing - NEW (much faster) does what
● Detects when new nucleotide is added to the chain
what is Pyrosequencing
sequencing by synthesis
● 1 million reads occur simultaneously
what is an activated nucleotide
nucleotide with 2 phosphate
groups added
what does apyrase do
degrades activated nucleotides which have not been added to the template strand (not complementary)
Process of high-throughput sequencing/pyrosequencing
- Single strand of DNA, sequencing primer,
enzymes (DNA polymerase, ATP sulfurylase,
luciferase, apyrase) and substrates (APS and
luciferin) and one of activated nucleotide (ATP,
TTP, CTP, GTP) all added - When a (complementary to template strand)
activated nucleotide added to strand of DNA - 2 phosphate groups released as pyrophosphate
(PPi) - APS + PPi → ATP (in presence of ATP sulfurylase)
- Luciferin → oxyluciferin (in presence of luciferase and ATP)
- Light is released
Process of high-throughput sequencing/pyrosequencing image
Uses of DNA sequencing
● Genome-wide comparisons between individuals and species
Sequence the DNA of a whole genome → determines the complete DNA sequence of
organism
what can we do using dna sequencing
Can analyse & compare genomes of many humans
● Epidemiology - the study of the distribution and determinants of diseases
● Reveals diseases to which individuals are vulnerable to & likelihood of developing
certain diseases
Can analyse genomes of pathogens
● Identify source of infection
● Identify antibiotic-resistant strains
● Track outbreaks of potential epidemics
Can search for evolutionary relationships between organisms
● Identify species
● Identify when 2 different species diverged
● Investigate genetic variation
o More differences in genome between individuals → more genetic variation
Can interpret genotype-phenotype relationships
● Stop expression (“knock out”) different genes → what effect does this have on the
organism’s phenotype
● Can target specific base sequences to stop the expression of certain genes
what is Bioinformatics
- development of software needed to analyse and store raw biological data in
a database
● Data such as: DNA/RNA/protein sequences
what is computational biology
- uses raw biological data to build theoretical models of biological
systems, using computational techniques
what can computational biology help us predict
Can predict what will happen in certain situations
Prediction of the sequences of amino acids in polypeptides
This means…
Can predict how protein folds into its tertiary structure
Can use this for synthetic biology
what is synthetic biology
Synthetic biology is the creation of new or redesigning existing biological systems and
molecules
● Large alterations to an organism’s genome → cells act in completely new ways
Application of synthetic biology:
Production of medicines
what does dna profiling determine
genetic identity (DNA characteristics)of an individual
Uses of DNA profiling:
● Paternity and maternity tests
● Proving/disproving family relationships
● Identifying species & evolutionary history of organism
● Identifying in forensics
● Identifying individual with high risk for particular disease
DNA Samples are compared to:
Saliva
Blood
Hair roots
Skin cells
The process of DNA profiling
- DNA obtained from individual
o Extracted from sample
o PCR used to give many copies of sample - DNA digested with restriction enzymes
o Restriction endonuclease enzymes cut the DNA at specific restriction sites
within introns
o DNA cut into fragments (vary in size between individuals)
o VNTRs (variable number tandem repeats) known as STRs (short tandem
repeats) found within introns
● Number of STRs varies from person to person - Fragments separated by gel electrophoresis
- Radioactive/fluorescent DNA probes added which act as markers
o Bind to complementary strands of DNA
o Different banding patterns can be seen - DNA to which the individual’s DNA is being compared with is treated with the same
restriction endonuclease enzymes & subjected to gel electrophoresis
what are we looking for in dna profiling
to have many fragments in common with the sample
Short, single-stranded RNA/DNA sequences complementary to known DNA
sequence
● Bind to complementary strands of DNA (is complementary to DNA which is being
investigated)
● Act as markers
o Labelled by radioactive or fluorescent markers
what are STRs (short tandem repeats)
- highly variable, short, repeating lengths of DNA (found in
introns - do not code for proteins)
The exact number of STRs vary from person to person
who are Short VNTRS (variable number tandem repeats) unique to
VNTRs unique to everyone, except identical twins