Manipulating genomes key processes

Seite 1

Method of DNA profiling:

EXTRACTION = DNA is extracted from the cells of the sample

DIGESTION = restriction endonucleases cut at specific sites within the INTRON of the DNA, resulting in multiple DNA fragments, of varying lengths (the varying lengths gives the unique genetic profiles of individuals)

SEPARATION (ELECTROPHORESIS) = the digested fragments from the sample are placed in a well of an electrophoresis agarose gel. An ELECTRIC CURRENT is applied, and the DNA fragments move towards the ANODE, from the cathode (DNA = -ve charge so attracted to anode). The fragments are therefore separated by SIZE, with the SMALLEST fragments moving the FURTHEST

SEPARATION (SOUTHERN BLOTTING) = the gel is immersed in an ALKALINE BUFFER SOLUTION, to separate the DNA double strands into SINGLE STRANDS. The single stranded DNA fragments are transferred from the gel, to a NYLON MEMBRANE.

HYDRIDISATION = radioactive/FLUORESCENT DNA probes are added in EXCESS to the DNA fragments on the nylon membrane. The DNA probes bind to COMPLEMENTARY DNA sequences (usually known as microsatellite regions)

DEVELOPMENT + VISUALISATION = if RADIOACTIVE probes were added, X-RAY images are taken of the nylon sheet. If FLUORESCENT probes were added, UV images are taken of the nylon sheet, to view the results

DNA must be made single stranded during electrophoresis BECAUSE: makes it easier to work with and analyse whilst profiling the DNS and allows the probes to complementarily bind to the DNA sequence in hybridisation/ the fluorescent dyes

Made single stranded using an ALKALINE BUFFER SOLUTION (may be in electrophoresis gel before you begin, and not just after!)

Probes are useful in locating specific DNA sequences - examples:

Locating a specific gene for use in genetic engineering

To identify the same gene in a variety of different genomes from different species, when conducting genome comparison studies

To identify presence/absence of a specific allele for a particular genetic disease/ that gives susceptibility for a particular condition

MICROARRAYS = applying specific DNA probes on a microarray, complementary to the sequence for mutated alleles for genetic diseases can highlight their presence

Seite 2

The Steps of PCR:

DENATURATION = the DNA sample is heated to 96 degrees. The kinetic energy provided breaks the hydrogen bonds between complementary base pairs, and results in the separation of the two strands

ANNEALING = the temperature is lowered to 55 degrees, which allows hydrogen bonds to form between the PRIMERS, and the complementary bases on the target region of the DNA strand (1 on each strand). *Specifically designed primers allow scientists to target a single, specific region of the genome*

EXTENSION = the temperature is increased to 72 degrees (the optimum for Taq Polymerase - obtained from heat-tolerant bacteria). Taq polymerase binds to the template strand and attaches complementary dNTP's to create the a newly synthesised strand.

CYCLE = the denature, annealing and extension cycle is repeated multiple times, to create the desired number of copies of the targeted fragment. The products are stored at 4 degrees once the cycle has finished

PCR takes place in a THERMAL CYCLER (a machine that carefully controls and changes the temperature at programmed timings, to trigger the different steps in PCR)

The amount of DNA increases EXPONENTIALLY eg. 1 ---> 2 ---> 4 ---> 8 ---> 16 ---> 32 ---> 64 etc

Work out by eg. amount of DNA after 6 cycles = 2^6 = 64

USE OF PCR TO SEPARATE PROTEINS = SDS can be added to give them all a negative charge - ensures that they will separate according to mass then, and not charge. If want to separate according to charge, don't add the SDS! - can be used to identify types of haemoglobin proteins for diagnosis of diseases eg. SICKLE CELL ANAEMIA

Seite 3

The steps of DNA sequencing (Sanger sequencing):

Reaction mixtures are placed in a THERMAL CYCLER and undergo the same cycling as for PCR

STE UP THE REACTION MIXTURE = 4 reaction mictures are set up. Each include different fluroscent dye-labelled terminator dNTP's (A, T, C, G), each being of a different colour, so each tube has a different coloured dye. Also contained in the reaction mixture = single stranded DNA to be sequenced, x1 primer, Taq polymerase, excess of regular dNTP's

DENATURE = the DNA sample is heated to 96 degrees. The kinetic energy provided breaks the hydrogen bonds between complementary base pairs and results in the separation of the two strands

ANNEALING = the temperature is lowered to 55 degrees, which allows hydrogen bonds to form between the primers and complementary bases of the target region

EXTENSION = The temperature is increased to 72 degrees (optimum to Taq Polymerase). Taq Polymerase binds to the template strand and attaches complementary dNTP's to create a newly synthesised strand. When a dye-labelled terminator dNTP is added, the extension is TERMINATED.

ELECTROPHORESIS = the fragments are placed in a gel and an electric current is applied. Fragments are separated by SIZE, with the smallest moving the FURTHEST

SEQUENCING = Either bases can be identified by using UV light to dectect the fluorescence on the gel, OR, its better to run each fragment length through a LASER, resulting in a CHROMATOGRAM of peaks, that are translated into the nucleotide sequence (the fluorescentl labelled terminator dNTP's at the end of each fragment are detected and recorded as a peak on the chromatogram

HIGHT THROUPUT PYROSEQUENSING = faster, lower cost, automated DNA sequencer, many samples can be analysed in one go ('high throughput')

Seite 4

Uses of DNA sequencing:

GENOMICS = the study of the structure, function and mapping of genomes

BIOINFORMATICS = the development of software and computing tools needed to analyse and organise raw biological data eg. developing computer modelling of molecular structures and making databases

COMPUTATIONAL BIOLOGY = the study of biology, using computational techniques, to analyse large amounts of sequencing data eg. to work out the 3D structure of proteins, identifying genes linked to genetic diseases and determining evolutionary relationships

Computational biology and bioinformatics can be used together to COMPARE GENOMES ('comparitive genomics')

For all comparisons, a database of 'normal' genes is required; generated using BIOINFORMATICS

REASONS FOR COMPARING GENOMES USING DNA SEQUENCING

Studying genotype-phenotype relationships (prediction of phenotypes by analysing genotypes)

Epidemiological studies (comparing genomes of people with a diseases eg. breast cancer with those who don't help to detect particular mutations that can be responsible for an increased risk of developing the disease)

Understanding evolutionary relationships (DNA sequences of different organisms can be compared and DNA mutation rates can be calculated - can calculate how long ago two species diverged from a common ancestor, allowing phylogenetic trees to be built)

Identifying new species (DNA BARCODING = by comparing the genome of species to be identified, with standard sequences of closely related species, particular sections of the genome can be compared that are common to all species, are highly conserved, but show SMALL VARIATIONS)

PROTEOMICS = the study + amino acid sequencing of an organism's entire protein complement

WHY CAN THERE BE MORE PROTEINS FORMED, THAN THE AMOUNT OF GENES WE HAVE?

RNA 'splicing' of mRNA, to splice out the introns = then, the exons are joined together, by enzyme complexes called SPLICEOSOMES. Not all the exons are included in the mature mRNA, giving rise to DIFFERENT PROTEINS (different combinations of exons can be joined together), so different phenotyopes. This is called ALTERNATIVE SPLICING!!!!

POST-TRANSLATIONAL PROTEIN MODIFICATION = different and more proteins can also be formed than egenes, due to post-translational protein modification. Eg. by phosphorylation, cAMP

SYNTHETIC BIOLOGY = the design and construction of novel biological pathways, organisms or devices, or the redesign of existing natural biological systems. Information from sequencing genomes allows biological molecules to be made from SCRATCH (synthetically), and then used to alter biological pathways

EXAMPLES OF SYNTHETIC BIOLOGY:

Genetic engineering-changing biological pathways

Industrial uses eg. use of synthetic immobilized enzymes, production of drugs from microorganisms

Synthesis of new genes to replace faulty genes eg. treatment of cystic fibrosis

Synthesis of a new organism eg. synthesising a bacterial cell by synthesising its genome