Manipulating Genomes Flashcards
What is Sanger sequencing ? (Chain termination method)
Uses modified nucleotides called DIDEOXYNUCLEOTIDES
- they pair with the complementary nucleotides on template strand during DNA replication
- when DNA polymerase encounters dideoxynucleotide on developing strand it stops replicating - hence CHAIN TERMINATING metho
Chain termination method ?
https://youtu.be/KRjfIXh_u2w?feature=shared
- 4 test tubes that contain the singe strand template DNA to be sequenced, DNA polymerase, DNA primers, free nucleotides A, C, T, and G, and 1/4 types of dideoxynucleotide; either A, C, T, or G
- Test tubes incubated at temp that allows DNA polymerase to function
- Primer anneals to the start (3’ end) of single stranded template - produce short section of double stranded DNA at start of sequence
- DNA polymerase attaches to double stranded section/begins DNA replication using free nucleotides in test tube
H bonds form between complementary bases on nucleotides - DNA polymerase can insert one of the dideoxynucleotides (ddNTP) by chance -> termination of DNA replication
- Complementary DNA chains produced vary in length , as when the ddNTP are inserted varies
- Once incubation period is over, the new complementary DNA chains separated from TEMPLATE DNA
- Single stranded DNA separated according to length using GEL ELECTROPHORESIS - smaller fragments travel further
What is high throughput sequencing ?
Describes multiple DNA sequencing technologies that allow simultaneous sequencing of multiple DNA strands
- faster /cheaper way to sequence genomes
Most methods are now AUTOMATED instead of manual interpretation - faster
E.g : capillary gel electrophoresis
Nanopore sequencing /pyrosequencing - next generation sequencing methods (newer)
What are high throughput sequencing methods useful for?
Fields fo study in:
Evolutionary biology /classfiication
Personalised med
Disease diagnosis
What is DNA Sequencing ?
Technique that allows the nucleotide base sequence an organism’s genetic material to be identified
What is a genome?
The complete set of genetic material present in a cell/organism
What is bioinformatics ? How has it allowed for comparison between genomes ?
Field of biology that involves the storage, retrieval, and analysis of data from biological studies
- studies may generate data on DNA /RNA /proetin sequences/relationship between genotype and phenotype
- large databases contain info about organisms gene sequences/ amino acid and protein sequences
- when genome is sequenced , bioinformatics allows scientists to make comparisons with the genomes of other organisms using the databases
- see how closely related organisms are
How is genetic variation and evolutionary relationships investigated by comparison of genomes?
Species with high level of genetic variation will have larger no. Differences in base sequences between individuals
Species with small no. Differences between their genomes likely to share more recent common ancestor than species with large no. Differences
- cytochrome C proetin can be used for comparison
How can genome sequencing aid the understanding of genotype-phenotype relationships?
Explored by stopping the expression of different genes /observing its effect on the phenotype
- when organisms genome sequence is known , scientist can target specific base sequences to ‘knock out’
Can also: predict an organism’s phenotype by analysing its genotype
Bioinformatics has allowed the comparison of all data and identified genotype-phenotype correlations - help predict what health problems people are at risk of
How can genome sequencing help in epidemiology?
Genomes of pathogens sequenced /analysed to aid research and disease control
- highly infectious strains can be identified
- The ability of a pathogen to infect multiple species can be investigated
- most appropriate control measures can be implemented/its spread
- Potential antigens for vaccine production can be identified
Computerised comparisons between genomes of those with and without disease used to detect any mutations that could pose increased risk of disease
How can gene sequencing be used to predict protein tertiary structure?
genetic code used to predict the amino acid sequence within a protein (order of bases codes for sequence of amino acids)
- can then predict how the new protein will fold into its tertiary structure
This information can be used in synthetic biology
What was the human genome project?
Began in 1990 - international,collaborative research programme
—> publicly funded so no commercial interests/influence
DNA samples taken from multiple people around the world, sequenced, and used to create a reference genome
Labs around the globe sequenced different sections of specific chromosomes
By 2003 the human genome sequenced to 99.9%accuracy
- finished genome was over 3 billion base pairs long but only about 25,000 genes (quite low)
Applications of the human genome project ?
- info from project used to tackle HUMAN HEALTH ISSUES - nd goal of finding CURES
- Scientists found a correlation between changes in specific genes and the likelihood of developing certain inherited diseases (cancers, Alzheimer’s )
What is a proteome and why is it difficult to determine the proteome for humans?
Proteome: full range of proteins produced by the genome
Large amounts of NON CODING DNA present in genomes
Regulatory genes/alternative splicing effect synthesis of proteins/gene expression
Why is the proteome larger than the genome?
Alternative splicing - allows for single gene to produce multiple proteins
Post translational modification of proteins
What is synthetic biology? What does it involve?
recent area of research that aims to create new biological parts, devices, and systems, or to redesign systems that already exist in nature
Involves : large alterations to organism’s genome
New genome can be made using existing DNA sequences or new sequences
- new sequences designed/written so they produce specific proteins
Well known use of synthetic biology? (Drug)
Production of ARTEMISININ - antimalaral drug
Scientists constructed a DNA sequence for a new metabolic pathway containing genes from BACTERIA, YEAST and A.annua- plant
- pathway results in production of ARTEMISINIC acid - precursor to artemisinin
Pathway can be inserted into yeast cells which will produce artemisinic acid
This precursor is converted to ARTEMISININ by inexpensive process
What is polymerase chain reaction (PCR) for?
In vitro method of DNA amplification
- to produce large quantities of specific fragments of DNA or RNA from very small quantities - for analysis
What does each PCR reaction contain?
Target DNA/RNA to be amplified
Primers (forward/ reverse)- short sequences of single-stranded DNA* complementary to the 3’ end of the DNA or RNA being copied.
DNA polymerase - enzyme used to build new DNA/RNA strand (normally TAQ POLYMERASE)
Free nucleotides - used in construction of DNA /RNA strands
Buffer solution - provide optimum pH for reactions to occur in
3 stages of PCR?
DENATURATION: the double-stranded DNA is heated to 95°C - breaks the H bonds between 2 strands
ANNEALING – temp decreased to 55°C so that primers (forward and reverse ones) can anneal by complementary base pairing to the ends of the single strands of DNA
ELONGATION/EXTENSION– temp increased to 72°C for at least a minute, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules
PCR occurs in thermal cycle - provides optimal temp at each stage/controls length of time at each stage
Can only amplify short sequences rather than whole chromosomes
Why is TAQ polymerase used in PCR?
- Comes from thermophilic bacterium - doesn’t denature at high temps
- It’s optimum temp is high enough to prevent annealing of DNA stands that have not been copied yet
- So that PCR cycle can continue repeatedly without stopping (to reload enzyme)
What is electrophoresis?
Process used to separate proteins or DNA fragments of different sizes
- molecules separated according to size/mass and their net charge
Why does electrophoresis work?
Electrical charge : + molecules will move towards CATHODE (-) and entire molecules mov towards ANODE (+)
- DNA is - charged bc of the phosphate - molecules move towards anode
Different sizes: tiny pores in gel mean smaller molecules move quickly /larger molecules move slower through the gel
Type of gel: different gel have different sized pores which affect sped at which molecules can move through them
How to prepare DNA for sequencing /analysing ?
First amplify (increase number) of DNA molecules by PCR
Method of electrophoresis ?
- Create an agarose gel plate in a tank. Wells are cut into the gel at one end
- Submerge the gel in an electrolyte solution (a salt solution that conducts electricity) in the tank
Insert the fragments into the wells using a micropipette - Apply an electrical current to the tank. The negative electrode must be connected to the end with the wells as DNA fragments will move towards the anode
- The smaller mass / shorter pieces of DNA fragments will move faster and further from the wells than the larger fragments
What are probes in electrophoresis?
Single stranded DNA sequences that are complementary to the VNTR regions
- contains a means by which to be identified:
A RADIOACTIVE LABEL: probes emit radiation that makes X-RAY film go dark, creating a pattern of dark bands
A FLUORESCENT STAIN/DYE : fluoresces when exposed to uv light - create pattern of coloured bands
How can gel electrophoresis be used to show genotypes?
By separating polypeptide chains produced by different alleles
How do scientists create a DNA profile?
- Obtain DNA - from root of hair, blood,semen, saliva
- Use PCR to produce large quantities of required fragment of DNA
- Use restriction endonucleases to cut amplified DNA molecules into fragments - cut close to target VNTRs
- Separate VNTRs using gel electrophoresis /alkaline is used to separate double strands of DNA
- Add radioactive/fluorescent probes that are complementary /bind (hybridise) to specific VNTR regions
- X-ray images produced /UV lights used to produce images of fluorescent labels - pattern of bars (DNA profile ) to be analysed
Use of DNA profiling?
Forensic med/criminal investigations :
Identify suspects for crimes
- Samples of body cells or fluids taken from the crime scene or victims body (eg. rape victims)
- DNA is removed and profiled
- The profile is compared to samples from the suspect , victim and people with no connection to the crime (control samples)
- Care must be taken to avoid contamination of the samples
- also used to identify bodies /body parts that are unidentifiable
- risk of developing particular diseases - certain VNTR associated with increased risk of cancers/heart disease
- determine familial relationships - who the father is/immigration cases (see if family are related)
- species conservation - reduce chances of inbreeding
Synthetic biology applications?
Info storage - encode large amounts of digital info onto single stand of synthetic DNA
Produce meds - genetic engendering used to produce ARTEMISININ
Novel proteins- designed proteins - e.g protein similar to Haemoglobin/bind to O2, not CO
Biosensors - modified bioluminescent bacteria placed on a coating of a microchip —> glow if air polluted
Nanotechnology- material produced for nanotechnology
Applications of gene sequencing?
Understanding evolutionary relationships/variation and comparison between species
Predict amino acid sequence of proteins
Synthetic biology
Epidemiology
Hybridise meaning?
Form H bonds with VNTR regions
Protein electrophoresis differences to DNA electrophoresis?
Used in diagnosis of med conditions where abnormal protein (caused by DNA mutation) is responsible for the disease (sickle cell anaemia)
- proteins need to be denatured first to pass through gel —> HEAT (t expose charges/hydrophobic regions)
- sodium dodecyl sulphate (SDS) added to make all proteins have negative charge to move through gel
Why is genetic engineering possible?
Geentic code is universal
- same codon code for same amino acids in all living things
Why is only non coding DNA used for DNA profiling ?
Contains VNTR regions
- using coding sequences wouldn’t provide unique profiles
2 differences between somatic gene therapy and germ line therapy?
Somatic Cannot be inherited / germ line can be
Gene is introduced into non-reproductive cell / introduced to gametes
Temporary /permanent
Target specific tissues in need of treatment / germ line cannot
What is germ line therapy?
Altering the alleles in sex cells
- any changes made to cells of parent will be inherited by offspring
Advantages of gene therapy?
Prolog lives of people with genetic disorders/give better quality of life
Baby of carriers of genetic disorder can be born without disorder/risk of cancer (in germ line)
Decrease no. Ppl that suffer genetic disorders (germ line)
Disadvantages of gene therapy?
- tech can be misused/used for things other than medical treatment (cosmetic effects etc)
- Can do more harm than good
- Expensive - resources cold be better spent on treatment that have passed clinical trials
- Effects of treatment can be short lived (somatic)
- May need multiple treatments (somatic)
- Difficult to get allele into specific body cells
- Body could have immune response against VECTORS
- Allele can be inserted in wrong place in DNA —> cancer
- Inserted allele could be overexpressed (too much of missing protein)
