MEDS2013 - Frontiers of Human Health

DNA bases

ATCG

RNA bases

AUGC

DNA

double stranded, more stable, stays in nucleus, has one job

RNA

single stranded, moves all around cell, has many jobs

DNA stays in the nucleus because

it is bulky and precious

transcription

small portion of DNA is copied into RNA

translation

ribosomes convert RNA code into amino acids

mRNA processing

introns removed, add 5’ cap and 3’ polyAtail to mRNA

introns

portion of DNA in a gene that is not expressed as a protein

exons

portion of DNA in a gene that is translated and expressed as a protein

3 nucleic acids is

a codon = amino acid

translation

tRNA brings amino acid to ribosome that reads mRNA sequence and links the amino acid into a polypeptide chain that folds into protein

gene

small piece of DNA that is transcribed as mRNA and translated as a protein

genome

all of the genetic material in an organism

histone proteins

DNA is wound around structural proteins that make up a nucleosomes and these are packaged into chromsomes

haploid

1 set, gametes

diploid

2 sets

why cells need to regulate transcription

no reason to express entire genome at all times where only a small portion of DNA is expressed as proteins

closed chromatin

heterochromatin is densely packed where transcription cannot occur

open chromatin

euchromatin is loosely packed where transcription can occur

allele

versions of a gene

diploid organism

have two copies of each allele

autosomal

alleles encoded on chromosomes 1-22

sex linked

alleles encoded on X/Y chromosome

autosomal traits

widows peak, earlobe attached, eye colour, hair colour

sex linked traits

colourblindness, haemophilia, webbed toes, ear hair

nucleotide composition

base, sugar, phosphate

DNA structure

chains antiparallel, sugar phosphate backbone on outside and base pairs inside

double helix has

wide major groove and narrow minor groove

RNA can form

secondary structures via base pairing

absorption spectrum for DNA

1 for 50ng/uL

absorption spectrum for RNA

1 for 40ng/uL

gene

DNA sequence that encodes products that carry out functions in the cell, can be protein coding or RNA coding

transcription

producing nucleotide-for-nucleotide copy of an RNA molecule from a DNA template

translation

producing a polypeptide chain from a mature mRNA transcript

two DNA strands in transcription

coding strand that codes for gene of interest and is the same as RNA produced and the template strand

mRNA in translation is read in

triplets (codons) to produce a polypeptide chain

the genetic code

used to translate RNA sequence into an amino acid sequence

standard number of amino acids in genetic code

20, 64 codons where 61 code for an amino acid and 3 serve as stop codons

Met (AUG)

start codon to initiate translation

structure of protein coding genes

RNA polymerase synthesises mRNA precursors (pre mRNA) where introns are removed to produce mature transcript comprised of exons

mutations within exons

affect protein structure/function

mutations within introns

affect protein expression as introns contain regulatory regions

mutations

alteration in the DNA that carries the genetic information

mutations only passed onto offspring fi it is in

germ cells

proteins

polymers made of amino acids using the genetic information stored in the genom

polypeptide chain

successive amino acids are joined togethered by peptide bonds

each codon represents

a particular amino acid

stop codons

3 codons signal the end of a polypeptide chain

first codon

start codon is AUG encoding methionine

open reading frame

any sequence of DNA or RNA beginning with a start codon and which can at least be translated into a protein

DNA is double stranded + each strand has 3 reading frames

→ a DNA strand has 6 possible reading frames in total

mutations on reading frame

single insertion (reading frame changed), three base insertion (reading frame not changed), nonsense mutation

DNA level mutations

base substitution, deletion, insertion, inversion, duplication, translocation

base substitution

one base is replaced by another base

deletion

one or more bases are deleted from the DNA sequence

insertion

one or more bases are inserted into the DNA sequence

inversion

a segment of DNA is inverted but remains at the same location

duplication

a segment of DNA is duplicated, the second copy usually remains near the same location as the original

translocation

a segment of DNA is transferred from its original location to another position either on the same DNA molecule or on a different DNA molecule

mutations at protein level

silent, missense, nonsense, frameshift

silent mutation

an alteration in the DNA sequence that does not change the amino acid being incorporated into the protein (synonymous mutation)

missense mutation

a change in the DNA sequence that results in a different amino acid being incorporated into the protein

nonsense mutation

a change in the DNA sequence that converts a codon into a premature stop codon

frameshift mutation

a change in the DNA sequence that is not a multiple of 3 and shifts the reading frame altering all downstream amino acids

null mutation effect

inactivates the gene

silent mutation effect

no effect on the amino acid, may or may not affect the phenotype

leaky mutation effect

partial activity remains

location of mutation determines

the effect of the mutation

mutations that affect the active site of a protein or structure of a protein

have the biggest effects

mutant proteins

sometimes be defective only under certain conditions - high temperature

DNA can be damaged by

radiation and by chemicals

spontaneous mutations occur due to

errors in DNA polymerase activity during DNA replication and spontaneous cytosine deamination

DNA rearrangements

due to mistaken pairing of similar sequences followed by recombination

karyotyping

a cytogenetic test that examines the number and structure of chromosomes, can only detect large-scale chromosomal changes like deletions, duplications and translocations, detects aneuploidy, stains chromosomes and visualise them under a microscope to identify structural and numerical changes

fluroescent in situ hybridisations

use fluorescently labelled DNA probes to identify specific DNA sequences on chromosomes, allows for the detection of smaller chromosomal abnormalities that might be missed by karyotyping

FISH identifies

deletions, duplications, translocations, and other structural or numerical abnormalities in chromosomes, determines the location of specific genes on chromosome

FISH use

cancer treatment for diagnosis, prognosis, predicting response for certain therapies

restriction digest

restriction enzymes recognise specific sequences within a DNA molecule and then make a double stranded cut

most recognition sites for restriction enzymes are

inverted repeats of 4,6 or 8 bases

type 1 restriction enzymes

cut the DNA at a random site more than 1000 base pairs from the recognition sequence

type 2 restriction enzymes

cut DNA within the recognition sequence, some generate blunt ends and others create sticky ends

restriction enzyme mapping

process of determining the locations of restriction enzyme recognition sites within a DNA molecule

restriction fragment length polymorphism

sample DNA is digested using specific restriction enzymes, fragments are analysed using gel electrophoresis to separate them based on their size

RFLP applications

detect genetic disease, identify a carrier of a disease-causing mutation, paternity tests, criminal investigations

polymerase chain reaction

amplify distinct DNA sequences, only requires tiny amounts of DNA, results in large amounts of identical copies, can add, delete and modify the DNA, uses DNA polymerase, specific primers complementary to the beginning and end point of the target sequence, 20-40 cycles

PCR applications

used for diagnosis to amplify and detect the presence of - genes associated with a genetic disorder, mutations in oncogenes, viral genome in the host cell, genetic sequence associated with a bacterial toxin, new mutant forms of viruses to help monitor the spread of diseases

sanger sequencing

determining the nucleotide sequence of small targeted region of DNA, chain termination sequencing, only one piece of template DNA, synthesis partial copies of a target DNA using DNA polymerase that vary in length by one nucleotide and then separating them by size, final nucleotide of each fragment labelled with a fluorescent tag, fragments separated by size

sanger sequencing application

detect mutations in specific genes associated with inherited disorders, identify bacterial species, determine drug resistant mutations in viral populations

protein level analysis

mass spectrometry to analyse the amino acid composition of proteins

mass spectrometry

analyse the mass-to-charge ratio of peptides derived from proteins, identify mutations that results from amino acid changes, can quantify the abundance of different peptides which can study the impact of mutations on protein expression levels or post-translation modifications

newborn heel prick screening

detect inborn errors in metabolism caused by mutations in genes involved in metabolism

electrophoresis

separate molecules based on their size and charge, can detect abnormal haemoglobin variants which can indicate protein mutations

gene expression

process by which information from a gene is used to synthesise a functional gene product most often a protein

transcription

DNA to RNA

translation

RNA to protein

gene expression is crucial for

cell differentiation and development, cellular function - different cell types have distinct functions and thus require different proteins, adaptation to changing environments

DNA accessibility

refers to the degree to which DNA is physically available for interaction with other molecules crucial for gene expression

DNA accessibility is a dynamic property

varies across the genome and even within the same genomic region across different cells or at different times

DNA is packaged into

chromatin