Classic genetic Technologies Flashcards
The structure of DNA
A nucleotide consists of:
A nitrogen base (A= T, G= C)
A sugar molecule (deoxyribose)
A phosphate group (negatively charged)
The characteristics of genetic code
It is made up of codons, triplets of bases. Each codon specifies a specific amino acid.
Three “stop” codons; UAA, UAG, UGA
There are 20 amino acids and 640 possible codon triplets.
mRNA reads 5’ to the 3’ end.
If there are mutations or error in the DNA, the message may be changed and incorrect protein formation results.
Types of restriction enzymes
Type 1 - cleave at a site that differs and is a random distance away, from their recognition site.
Type 2 - cleave within or at short specific distances from a recognition site (most useful in laboratory)
Type 3 - cleave at sites a short distance from a recognition site
Type 4 - target modified DNA, e.g. methylated hydroxymethylated and glucosyl-hydroxymethylated DNA.
Type 5 - utilise guide RNAs to target specific non-palindromic sequences (used in CRISPR-cas9 technology)
Gel electrophoresis
DNA - naturally occurring negative charges carried by their sugar phosphate backbone
When DNA is loaded onto a Greek and an electric field applied - the DNA molecule with move towards the electrode
The DNA fragments will separate according to size
What are the types of Gel
Agarose - most commonly used in the lab
Polyacrylamide - separation of small DNA fragments or fragments that have small differences in size
The percentage of gel depends on fragment size to be analysed - small size, higher gel percentage
Staining is the most common way to visualise DNA
Southern blotting
Transfer DNA fragments directly from gel onto a nitrocellulose membrane laid on top of it, providing a semi-permanent copy of the electrophoresis pattern and allow further investigation of the DNA
Autoradiography of the membrane would reveal the specific fragments containing the sequence of interest, which would appear as sharp bands in the same position as they had been on the gel.
It was then possible to detect a specific DNA sequence from a smear, without having to purify it away from the rest of the genome.
Other strategies for different molecules.
RFLP mapping
(Restriction fragment length polymorphism)
RFLP
- variation between individuals sequence leads to a change in a restriction enzyme site, and a characteristic banding pattern occurs when DNA probe is hybridised to the chromosomal DNA in a southern blot analysis
Confirms the existence of polymorphic variation in DNA sequence between individuals
Genetic linkage
Tendency of DNA sequences, close together on a chromosome, to be interred together.
Two genetic markers that are physically near to each other are unlikely to be separated onto different chromatids during chromosomal crossover, and are therefore said to be more linked than markets that are far apart.
Markets on different chromosomes are perfectly unlinked.
A linkage map shows the position of it’s known genes or genetic markers relative to each other in terms of recombination frequency, rather than a specific physical distance.
Parametric Linkage analysis
Analysis may be:
Parametric
- mode of inheritance and penetrance are defined.
- assesses the probability that a gene important for a disease is linked to a genetic markers is studied through the LOD score
- a LOD score of 3 or higher means the odds are a thousand to one that the two genes gene and markers are linked, are and are therefore inherited together.
Typically employed for single gene disorders and Mendelian forms of complex disorders.
Non-parametric linkage analysis
Non-parametric
- evaluate whether segregation at specific locations is “not-random”, specifically, the objective is to show increased IBD (identical by descent) sharing among sets of affected individuals
- no assumptions are made about the disease model
-ASP (affected-Sib-Pair test) is the simplest form
1. Pairs of phonetically similar sibs will tend toward excess sharing of relevant chromosomal regions.
2. Dissimilar sibs will tend towards lower sharing
3. Ascertain a set of sib pairs (concordant or discordant) for a given trait
4. Assess the degree of intrapair genetic similarity at locus by estimating the sharing pattern, the probabilities that a typical pair shares, zero, one or two alleles identical by descent
5. Evaluate statistical significance
Used to study more complex, common traits
Positional cloning
Gene for a specific phenotype is identified only by its approximate chromosomal location defined using linkage analysis
Positional cloning is then used to narrow the candidate region until the gene and its mutations are found
Process = chromosome
(DBA fragments from the coldest known genetic markers are progressively cloned and sequenced, getting closer to the mutant allele with each new clone)
Has been used to identify:
- Huntingtons disease
- cystic fibrosis
Sanger sequencing
Dideoxy-nucleotides (ddNTPs), lack a 3’-hydroxyl abs therefore cannot be extended by DNA polymerase i.e terminates the in vitro extension of the DNA.
Set up 4 DNA synthesis reactions containing:
- The same ssDNA template
- primer
- DNApolymerase
- normal deoxynucleotidse (dNTP)
AND - one radio labelled ddNTP and dNTP are present, DNA polymerase would sometimes incorporate the correct dNTP, allowing further polymerisation, and at other times, would increase the ddNTP, causing chain termination
Polyacrylamide gel electrophoresis to separate the synthesised fragments
Polymerase chain reaction (PCR)
PCR amplifies (copies) a specific region of a DNA strand —> PCR product is used for downstream analysis
- sizing
- sequencing
- RE suggest to detect SNVs
PCR relies on t thermal cycling —> exposing the reactants to cycles repeated heating and cooling, permitting different temperature- dependent reactions specifically:
- DNA denaturation
- primer annealing
- Elongation
Human genome project
Aim-determine the DNA sequence of the entire euchromatic human genome within 15 years
92% of the genome is sequenced
‘BAC based shotgun’ sequencing
The ‘modern’ diagnostic laboratory
Most sequencing - NGS/WGS
Sanger sequencing remains the golds standard
Fragment analysis (PCR based sizing methods) still currently required to confirm some sizes of triplets repeat disorders
Southern blotting - rare but still necessary to detect large scale variation
The future of genetic testing
Centralised sequencing hubs
Centralised data repositories
Fragment analysis and gene dosage possible using WGS data
Huge potential
- discover new genes, genes involved in complex disorders, and the prospect of personalised medicine which is already a reality for some cancer syndrome
- 3rd generation sequencing
