polymorphisms and mutations

why use DNA as a diagnostic tool

• dna profiling

• dna fingerprinting

• diagosting tests: Diagnose genetic variant underlying a disease or phenotype

  Determine individuals who may be pre-disposed to developing a genetic disorder

  Enable the selection of desirable traits in the livestock, or the breeding out of less desirable traits

what does the choice of test depend on

• isthe underlying mutation known or not

• what size is the lesion- point mutation, whole chromosome?

Klinefelter syndrome

• XXY- extra sex chromosome

• individuals often have no phenoytpe but can cause sterility of hypogonadism

karotying

• what is it used for

• what stage must chromosomes be

used for detection of gross changes in number orsructure of chromosomes

chromosomes must be in metaphase to discern by eye as above

karotyping-chromosomal defects

• what are many cancers caused by and what does this form

• examples

• many cancers are caused by chromosomes breaking and reattaching to other chromosomes forming fusion genes eg chronic myelogenous leukaemia

hi res karyotyping

• what can this distinguish and why is this a disadvantage

• how much percent of variation in the human genome come from copy number variations

• can only distinguish large deletions or duplications (>3Mb DNA)

• yet many deletions that cause DNA are smaller (Kb rather than Mb)

• 13 percent of variation in human genome comes from copy number variations- small diffs in the copies of DNA sequences between individuals

array comparative genomic hybridisation

1. sample and normal reference dna labelled and hybridised to chip spotted with bits of every chromosome

2. if there are equal numbers of copies of a DNA sequence then the spot goes yellow. if there are more copies of a sequence in the sample than in the reference then the signal is biased towards the sample and goes red. if there are fewer copies in the samplle DNA than in the normal DNA then signal is biased towards the normal and goes green

3. slide scanned to quantify amounts of red and green signal

4. plot of gain or loss of DNA along chromosome generated.

array CGH readouts

pre implantation genetic diagnosis

• performed in conjunction with ivf from parents with genetic disorder

• multiple embryos made from parents egg and sperm

• 1-2 cells removed from each

• dna isolated and tested for mutation/ chromosomal abnormality

• embryos without the mutation implanted into mothers uterus

• embryos with the mutation are discarded

• screen single gene mutations, karyotype changes, or copy number variations

VNTR polymorphisms and DNA fingerprinting

• how much base pairs and how many times repeated for mini vs micro

• what can we use these differences for

• genome contains large numbers of mini or micro satellite repeat sequences- VNTR

• •Mini: 10-100bp in length, repeated >1000 times (e.g. at telomeres)

  •Micro: 2-10bp in length, repeated 4-40 times

  •Variable between individuals

  •Utilise these differences to identify individuals or parentage

performing a dna profile with VNTR

•Isolate DNA from samples of interest

•Break into small fragments (restriction enzymes)

•Run on gel to separate by size (electrophoresis)

•Blot gel onto membrane (Southern Blot)

•Incubate with radioactive probe that binds VNTR sequences only

•Image blot on photographic film – only areas where probe has bound will expose onto film, giving bands

restriction fragment length polymorphisms

• utilises restriction enzymes which recognise specific dna sequences and cleave dna at or near the recognition site

limitations of using rflps for diagnostics for

• designing PCR primers

• selecting an appropriate restriction enzyme

• mutation location

• need to know the dna sequence either side of the mutation site to design pcr primers

• need to know the mutation in order to select an appropriate restriction enzyme

• mutation needs to be in a restriction site

• different asay required for each mutation

sanger method

• oh group allows bonding to next base in chain-dNTP

• h group in place of oh inhibits bonding so stop any more nucleotides from being added- ddNTP

sanger

Perform PCR with mix of dNTPs and ddNTPs – when ddNTP incorporated, chain elongation stops

Creates lots of molecules of different lengths – separate on gel to determine sizes. 

By linking different coloured flourescent dyes to each ddNTP, we can determine what nucleoetide is at the end of the chain.

identifying nucleotide variations by sanger

• isolate dna from individual

• perform pcr for region of interest

• sequence pcr product

limiting of the sanger sequencing

• can only read 500bp accurately which is equivalent to 2-3 exons for most genes. problem for large genes

• error prone- 1/1000 called incorrectly. need to sequence repeatedly to ensure correct seq.

next gen sequencing approaches

• exome sequencing- sequence all exons

• transcriptome sequencing (rna-seq)- sequence all rna in a cell- mrna trna, non coding rna

• whole genome sequencing- sequence the entire dna sequence of the individual

limitations of next gen

• huge amounts of data

• need special software and users to interpret data correctly

• big variation between individuals

• exome sequencing only idnetifies variants in protein coding sequences- splice site mutations, non coding dna variants?

nanopore sequencing device

• a small disposable sequencing chip tha t contains the nanopores, required fluidics and eectronic sensors

• only need prepared dna sample and a laptop with usb to plut