6.3 Manipulating genomes Flashcards
define genome
the complete genetic make up of an organism
define DNA sequencing
a technique allowing genes to be isolated and read
what did early DNA research show
structure of DNA was known
base triplet sequences coded for amino acids
but you couldn’t work out the sequence of nucleotide base triplets
explain Fred Sanger’s DNA sequencing approach
use a single strand of DNA as a template for 4 experiments in seperate dishes containing
one of the 4 different terminators used (modified nucleotides of ATCG)
normal nucleotides
a primer
DNA polymerase
DNA fragments of various lengths were made and pass through a gel by electrophoresis
sorted by length (shorter travelled further)
bases get read off what terminator it had and it creates the sequence
explain how DNA gets cloned
gene isolated using enzymes from bacteria
inserted into bacterial plasmid (vector) and then into an E coli bacterium host
when cultured divided lots
plasmid in DNA copied many times
each new bacteria contains DNA
what technique is used in high throughput sequencing to sequence genomes
pyrosequencing
what is pyrosequencing
synthesisng a single strand of DNA complementory to the strand to be sequenced, by one base at a time whilst detecting the light admission
explain the process of pyrosequencing
DNA cut into fragments using a nebuliser
lengths are degraded into single strands of DNA
sequencing primer is added
DNA is incubated with DNA polymerase, ATP sulfurylase, luciferase, apyrase, luciferin and APS
activated nucleotides (TTP, ATP, CTP, GTP) are added one at a time
light detected
what is the human genome project
project launched to sequence the human genome
contains about 24000 genes
how can genome wide comparisons be made between species
whole genome sequencing determins the complete DNA sequence of an organisms genome
how can genome wide comparisons be made between individuals
all have same genes but different alleles
mutations can cause difference between DNA sequences
what is bioinformatics
developing and using computer software that can analyse / store biological data
how can we predict the amino acid sequence of proteins
can work out the base triplets which code for an amino acid = can determine the orimary structure of an amino acid
define intron
non coding bit of DNA that doesn’t code for an amino acid
define gene
sectionof DNA that codes for a protein and results in a characteristic
define locus
position of a gene on a chromosome
what is synthetic biology
involves designign and building biological dveices and systems
uses of synthetic biology
information storage onto single strands of DNA
productionof medicines
development of porotiens - eg. haemoglobin
biosensors
nanotechnology
what is DNA profiling
technique used to determin patterns in the non coding DNA of an individual
explain the process of DNA profiling
DNA is obtained from the individual
digested with enzymes into fragments
fragments are sperated by gel electrophoresis
a banding pattern is created and can be compared
who developed DNA profiling
Alac Jeffreys
uses of DNA profiling
forensic science
maternity and paternity disputes
analysis of disease
define polymerase chain reaction
technology that can amplify a short length of DNA to lots of copies
process of PCR
DNA mixed with DNA nucleotides, primers, magnesium ions and enzyme Taq DNA polymerase
heated to 95 degrees c - breaks hydrogen bonds between complementary nucleotide base pairs into two single strands
cooled to 68 degrees c - primers can bind to one end of each single strand (gives a small section of a double strand)
DNA polymerase enzymes bind to the end where the primer is
heated to 72 degrees c - optimum temp for DNA poly
DNA poly catalyses nucleotides joining the single strand in the 5’ to 3’ direction
new double strand generated
repeats cycles
uses of PCR
forensics - small quantities of DNA can be amplified
analysis of disease risk - detecting mutations
identify viral infections
tissue typing - reduces risk of rejection of transplants
detection of cancer
research of extinct species
define electrophoresis
process used to seperate proteins/ DNA fragments of different sizes
explain how electrophoresis works
extraction of DNA from sample
fragments are created using restriction endonucleas enzymes)
seperation of fragments by running electric current over it (attracted to positive electrode at different rates)
in an agarose gel and buffer solution
smaller fragments = travel further
fluorescent dye/ probes used to show
how can electrophoresis be used for seperating proteins
charged solution to make all proteins have same charge
proteins seperate as they move through the gel
according to molecular mass
what is a DNA probe
short single stranded length of DNA complementory to section of DNA being investigated
has fluorescent dye/ radioactive tag
explain how a DNA probe is used
DNA probe binds to the
complemenotry section of the target DNA
the fuorescent dye tag is shown up when UV light passes over/ radioactive tag shows up dark when exposed to photographic film
identifies presence/ location of the traget DNA sequence
uses of DNA probes
locate specific gene
identify same gene in variety of genomes
idenitfy presence of an allele
what is genetic engineering
recombination DNA technology
combining DNA from different organisms by isolating them and inserting them into another organism using vectors
name the stages of DNA engineering
obtaining required gene
placing gene into a vector
get vector into recipient cell
recipient expresses gene
how is the required gene obtained in genetic engineering
mRNA from cells where gene is expressed is obtained
enzyme reverse transcriptase catalyses formation of single strand of complementary DNA
primers and DNA poly. added = makes into a double strand which codes for specific protein
DNA probe used to locate a gene = cut out using restriction enzymes
alternative ways to obtain gene in genetic engineering
nucleotide sequence known - gene synthesised using an automated polynucleotide synthesiser
or PCR primers to amplify the gene
explain how genes are placed into a vector in genetic engineering
plasmids from bacteria are mixed with restriction enzymes = cuts plasmid at specific recognition sites
cut ends are sticky ends (unpaired)
nucleotide bases are added = complementary to sticky ends of plasmid
DNA ligase catalyses annealing (binding) of gene and cut plasmid
alternative way to place gene into vector
gene placed into attenuated virus and carry it into host cell
explain the ways that vectors are placed into recipient cell
heat shock treatment - making walls/ membranes of bacteria more porous
electroporation - high voltage pulse applied to cell to disrupt the membrane
electrofusion - electrical fields used to introduce DNA into cells
Transfection - DNA packaged into bacteriophage , transfects the host cell
gene placed in attenuated v. carried into host cell
gene gun - small pieces of gold used, coated in DNA, fired into plant cells
define restriction endonuclease enzymes
cut DNA at certain recognition points
define vector
carry/ insert DNA into host organism
define sticky ends
single stranded ends of DNA that has been created by endonuclease enzymes
DNA ligase
joins DNA fragments
joins sugar and phosphate groups of DNA backbone
ethical issues with genetic manipulation
microorgansims
-escape, antibiotic resistane
- pathogens/virus may be inserted into genome which increases risk of cancer
plants
- super weeds created eg. resistance of herbicides in soya and weeds getting destroyed
- concerns about eating genetically modified food
- no choice whether to but gm seeds
pharming
- using genetically modified animals to make human proteins
Somatic cell gene therapy
Gene therapy by inserting alleles into body cells
Doesn’t pass to offspring
Germ line therapy
Gene therapy by inserting alleles into gametes/zygotes
Offspring may inherit gene
basic principle of gene therapy
Inserting a functional allele of a particular gene into cells containing mutated/non functioning alleles of the gene
How can a liposome be is used to insert genes
Alleles are packaged into the liposome ‘sphere of lipid bilayer’
Sprayed by an aerosol into the nose
Can pass into cells lining respiratory tract
Pass through plasma and nuclear membrane
Into genome - gets expressed
Epithelial cells are replaced every 10-14 days
issues with using viruses as a vector for gene therapy
May cause immune response
May become immune to virus making deliveries impossible
May insert gene in location that disrupts other genes involved in regulating cell division - increases cancer risk
Disrupts expression of other genes
