Molecular Genetics

What are SNP’s

• tiny differences in DNA between people

• stands for single nucleotide polymorphism

• theyre one reason people arent genetically identical

What is GBS

• genotyping by sequencing

• technique used to find lots of SNP’s by sequencing DNA

• cut up dna, sequence parts, compare individuals, identify SNP’s

Compare GBS and SNP array

• SNP is the genetic difference whereas GBS is the method used to find the genetic difference

• both GBS and SNP arrays are able to identify SNP’s but they use different SNP’s to do so

what are the advantages and disadvantages of SNP arrays

what are the advantages and disadvantages of GBS

What type of inheritance do mitochondrial DNA and Y chromosome show

• uniparental inheritance

• mitochondrial DNA only inherited along maternal line

• Y chromosome inherited

• mitochondrial dna does not recombine

what is the recombination like in autosomes and mitchondrial DNA + Y chromosome

• autosomes - recombine creates unique combination of SSR’s and SNP’s

• mito DNA and Y chromosome - do not recombine - dna sequence only changes through mutation

what are some features of the mitochondrial genome

• high copy number

• easy amplification

• lack of recombination

• maternal inheritance

• high mutation rate - particularly in non coding control region - D loop

• sequence contains SNP’s and microsatellites

what are the features of Y chromosome

• sequence contains SNP’s and microsatellites

• Y chromosome is single copy and has high content of repetitive DNA, more difficult to analyse than mitochondrial DNA

what is a haplogroup

• describes line of descent

• a group of people that share a common genetic ancestor because they all inherit the same genetic marker

• example - if theres a mutation and it gets passed down through generations everyone who inherits it can be grouped together in same haplogroup

• they are usually identified using the mito DNA and Y chromosome

How do you know if theres a mutation in your mito DNA

• need a reference sequence

• haplogroup analysis

• nucleotide changes

how to build and read a haplogroup phylogeny

• the big box H is a haplogroup

• further down you go the more specific the genetic group becomes

to build it:

• they look at peoples mtDNA and compare it with reference sequence - rCRS, then look for mutations

• same mutations = same genetic group

• the numbers are positions on the mtDNA where mutation has occured

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what is the difference between genomes in prokaryotes and eukaryotes

Prokaryotes:

• single chromosome and plasmids

• genes dont contain introns

• very little repetitive DNA

• genes often found in clusters

Eukaryotes:

• genomes have multiple chromosomes

• no plasmids

• organelle genomes

• genomes contain introns

• gene clusters rare but sometimes found

whats the difference between transcriptomes and genomes

• transcriptomes are smaller than genomes

• vary in size less as no repetitive sequence

what are the main steps to next generation sequencing

1. DNA or RNA extraction

2. library preparation

3. cluster generation

4. sequencing

what are the main steps in illumina DNA sequencing

• DNA is broken down into small fragments

• adapters and barcodes are attatched to each fragment

• adapters allow flow cells to be stuck to a flow cell and barcodes link the DNA fragment to the sample

• primers bind to the adapters

• each fragment is copied many times - this group of copies = a cluster

• this happens at the same time across the flow cell

What are the main steps in next generation sequencing

• machine supplies dNTP’s - each base has a fluroescent dye

• only one base is added at a time - temporary OH cap stops another base being added immediately after

• machine shines light on the DNA and records the fluorescent colour - can identify which base was added

• fluorescent dye and cap are removed - allows next base to be added

• machine repeats the process many times

difference between sanger and next generation sequencing

what is long read sequencing

• next next generation sequencing

• generates reads up to 1Mb which can span repeats and allow resolution of complex genomic regions such as centromeres and telomeres

• SMRT and oxford nanopore sequencing are two of the leading technologies

what is long read sequencing

• reads longers stretches of DNA in one go rather than breaking information into short reads

• long read sequencing technologies - PAC bio and oxford nanopore

whats PAC bio

• reads DNA by detecting flashes of light

• circular DNA is immobilised in small wells ready for sequencing

• uses DNA polymerase to add fluroscently labelled nucleotides to template

• sequencing is error prone so each templates sequenced multiple times

what is oxford nanopore

• motor proteins unwind dna and feed single stranded dna into a nanopore

• electrical current is passed over nanopore dna moves through pore