Mutations

Mutation

permanent change in DNA sequence
change in gene or chromosome leading to new characteristics in an organism

Induced mutations

mutation caused by mutagenic agent

spontaneous mutation

mutation that occurs due to an error in a natural biological process

Mutagens/mutagenic agents

factors that increase the rate at which mutations occur
e.g.

• viruses

• radiation (UV rays)

• microorganisms

• environmental poisons and irritants

• alcohol and diet

Cell division

• causes mutations (chromosomal) when chromosomes fail to separate/break apart

• specifiable during crossing over (break apart and reattach incorrectly) and non-disjunction (failure to separate)

DNA replication

DNA is copied during DNA replication pairs match based on complimentary base pairing rules (AT and CG)

If there is mismatching or nucleotides are added/removed incorrectly, it will lead to mutations (gene/point)

phenotype

observable characteristics due to the genotype

genotype

combination of alleles for a gene

Evolution

gradual change in characteristics of a species

Gene Mutations

a permanent alteration to a single gene

• affects single gene (loci) changing one base pair/thousands of base pairs • changes in a single gene so that the traits normally produced by that gene are changed/destroyed

Examples of gene mutations •       Sickle cell anaemia – substitution •       Cystic fibrosis – deletion •       Crohn’s disease - insertion

Chromosomal mutation

permanent change to the structure and/or number of chromosomes in an organism

• affect more than one gene up to a whole chromosome • all part of a chromosome is affected

Examples of chromosome mutations •       Cri-du Chat – Deletion •       Charcot-Marie-Tooth Disease – duplication •       Haemophilia A – inversion •       Robertsonian Translocation – translocation •       Aneuploidy etc caused by non-disjunction include Down Syndrome, Turner syndrome etc.

Somatic mutations

Not inherited by offspring

·       Occurs in all body cells except gametes

·       Will only affect certain cells (not all)

·       Diseases such as cancer

Germline Mutations

  May be inherited by offspring

·       Occur in gametes only

·       Will affect all cells in the body of the offspring

·       Often doesn’t affect the individual who has mutation

·       Diseases such as Huntington’s disease

heritability mutations

somatic mutations and germline mutations

extent of mutations

gene mutations and chromosomal mutations

Gene/point mutations

Insertion: nucleotide is added

Substitution: an existing nucleotide is replaced with a new nucleotide

Deletion: a nucleotide is removed

Chromosomal mutations (mechanical process)

Duplication(insertion): section of a chromosome occurs twice
Deletion: piece of DNA is removed
Inversion: chromosome breaks and reattaches the wrong way around
Translocation: chromosome breaks and reattaches to the wrong chromosome
Non-dysjunction: chromosome pairs don’t separate during meiosis

point mutations

specific type of gene mutation: insertion, deletion and substitution mutations as they only affect a single nucleotide (change in a single nucleotide therefore only one base is changed)

Frameshift mutation

where the amino acids after the insertion or deletion are all changed
- insertion and deletion may result in a frameshift mutation

WHY IS NON-DISJUNCTION in meiosis I result in a higher proportion of faulty gametes than non-disjunction in meiosis II?

• Because it occurs in the first division of meiosis which is crucial in the formation the egg cell

• in this division, homologous chromosomes are paired and separated into 2 cells

• If non-disjunction occurs, both homologous pairs end up in the same cell resulting in aneuploidy, leading to and extra chromosome (n+1) or missing chromosome (n-1) in the egg cell

• If it occurs in meiosis II, it doesn’t affect majority of the gametes as only half the gametes are produced during this division

aneuploidy

type of mutation where there’s a change in the chromosome number as a result of non-disjunction

non-dysjunction in meiosis

• when homologous chromosomes fail to separate properly in meiosis I, or sister chromatids fail to separate in meiosis II, causing one gamete to receive two of the same type of chromosome and the other gamete receives no copy.

• this results in abnormal numbers of chromosomes in the gametes

Gel electrophoresis

used to separate DNA strands (RNA or proteins) based on size
before running a PCR product through GEP, you must first use restriction enzymes to cut it to specific lengths/specific nucleotide sequences

GEP is used to create DNA profiles for identification of:

• individuals (forensics, crime, fossils)

• relatedness (ancestry, family relatedness, species identification)

• Genetic diseases - a mutant (disease) allele is often longer/shorter than a normal allele. Can be identified by comparing with the normal allele

GEP process

1. Prepare the Gel

2. Load the DNA samples

3. Apply an electric current

4. Document the results

Loading dye

glycerol - fat/thick which is dense, and sinks when injected into well, makes DNA visible

visualising dye

coloured to see dye going to well, see them under UV light (flourescence)

Description on preparing the gel

• the gel is made of a polymer, such as agarose, which is dissolved in a buffer • the buffer contains ions that allows electrical current to travel through the gel • Maintains a stable pH to allow DNA to separate appropriately • the conc. of agarose determines the size of the pores in the gel • smaller pores will separate smaller DNA fragments, while larger pores will separate larger DNA fragments

Description on loading the DNA samples

• the DNA samples are loaded into the wells using a micropipette

• the DNA samples will contain a loading dye and a visualising dye

• Loading dye makes it denser, so it sinks to the bottom of the well making loading easier

• the visualising dye will contain a fluorescent dye to enable it to be visualised under IV light

Description on applying an electric current

• An electric current is applied to the gel which causes the DNA fragments to move towards the positive electrode

• the DNA fragments will move through the gel at a rate that is inversely proportional to their size

• smaller DNA fragments will move faster than larger DNA fragments

Description on documenting the results

• the results of the gel electrophoresis can be documented by taking a picture of the gel or by scanning the gel into a computer

• the size of DNA fragments can be determined by comparing the positions of the bands on the gel to a DNA ladder

DNA sequencing

the process of determining the precise order of the nucleotides in a sample of DNA molecule

DNA synthesised from 4 nucleotides, each with different nitrogenous base - adenine, cytosine, guanine and thymine

• Nucleotides = deoxynucleotide triphosphate (contain 3 phosphate groups joined to sugar deoxyribose with its base)

What happens when DNA forms?

• each nucleotide loses two phosphate groups

• sugar molecule loses hydrogen atom from hydroxy group (OH) when it bonds to the phosphate group of an adjacent nucleotide

• synthetic nucleotide stops elongation of sequence because there is no OH group for the next nucleotide to attach to. This occurs to each nucleotide in the DNA sample, creating different lengths of DNA. These can be separated using gel electrophoresis. Knowing which base was added to create each length helps determine the order of nucleotides

purpose of DNA ladder

• help show length of different bandings

• help estimate size/no. of base pairs of DNA

Evolutionary biology

DNA sequencing can be used to compare the genomes of a different species, which can help us understand the evolutionary pathways

Medical diagnostic

DNA sequencing can be used to diagnose genetic diseases such as, cystic fibrosis, sickle cell anaemia etc., it can also be used to identify the risk of developing certain diseases such as cancer.

Understanding order of nucleotide sequencing allows us to:

• identify the degree of genetic variations in a population

• identify the degree of genetic diversity between species

• establish similarity between species

• quantify the degree of genetics molecular clocks/determine the time since organisms shared a common ancestor

  • genetics molecular clocks: technique used to estimate the time where different species diverged from a common ancestor

• Helps construct phylogenetic trees

• identify how genes derived from common ancestors have changed over time

• uses comparative genomics to help understand the process of evolutionary changes over time

Ethical considerations with genetic information

• Autonomy: respect for the right to be self-determining and choose whether or not to be tested. If tested to know and share the information, including the right of an individual to decide their own future, independent of genetic information

• confidentiality: use of genetic information is treated sensitively and is accessed only by those who are authorised to access it

• equity: right to fair and equal treatment regardless of genetic information

• Privacy: right to be ‘left alone’ and to make decisions regarding genetic testing and the resulting information independent for others

PCR (Polymerase Chain Reaction)

polymerase chain reaction uses cycles of heating and cooling and DNA polymerase (enzyme) to rapidly replicate DNA

purpose of PCR

amplify small amounts (degraded) of DNA to enable them to be studied and compared

PCR process

1. Template DNA - will contain all DNA from the sample

2. DNA primer two (forward and reverse) single stranded sequences that target the specific sequence to be copied

   • dictate what will be amplified

   • only sequences between the forward and reverse primers will be copied

3. DNA polymerase - (taq polymerase - simplifies it and automates and is thermostable) used to join dNTP’s to the template DNA)

4. dNTP’s are free nucleotides that will eventually form the new strands of DNA

5. Buffer solution - maintains the pH to enable optimal enzyme function

   • enzymes are sensitive to temp, pH

• temp changes controlled by thermocycler - which rapidly heat and cool PCR products

Denaturing

96 degrees celsius - used to break down hydrogen bonds holding the two strands

enzyme helicase separates two strands of DNA, allowing each strand to be replicated

• Heats the DNA to separate the double strand into single strands (denaturing)

• this provides a template for the annealing process
  

Annealing

55-65 degrees celsius

allows short strands of DNA called primers to bind to single DNA strands

• primers are complementary to either end of the sections of DNA to be copied

• Cools the reaction down, which allows specific primers to bind to complimentary sequence on the single stranded template strand

Extension

72 degree celsius

• Heats the reaction again to allow taq polymerase to add/join free nucleotides and extend the primers

• this results in the production of two identical double stranded DNA

Reliability

the extent to which an experiment gives the same results each time it is performed under the same conditions (consistent)

accuracy

extent to which the measurements are correct/close to true value
only possible is you’re using the same equipment

validity

extent to which an experiment tests what it’s supposed to test

how is reliability high/not low?

• using modern (new) - maintains voltage/current baseline throughout the entire run (20 mins)

• good (not contaminated) buffer solution) - right conc, won’t conduct too much electrical current preventing DNA fragments from denaturing

how to ensure reliability

• repeat trial on another day with the same gel and buffer solution

• run duplicate lanes → sample a, b and c have two lanes each sid by side to prove they have the same band positions

how to ensure accuracy?

• select right molecular ladder - ladder must have known sizes to frame unknown samples

• prevent sample overloading - if too much DNA is loaded it causes bands to look advanced/smeared

• don’t manually guess band size by eye but do it by using a pixel-analysis software