Variation and evolutions

How do dideoxynucleotide triphosphate (ddNTP) differ from normal dNTP’s?

Lack a hydroxyl group needed for further attachments. Therefore, when added to a forming strand of DNA, elongation of that strand is ended. The next deoxynucleotide is not able to bond onto the strand.

Requirements for DNA sequencing

• single stranded DNA

• Free floating nucleotides

• ddNTP labelled with different coloured florescent dye for easy detection

• RNA primers

• taq DNA polymerase

• DNA sequencing machine

DNA sequencing steps

1. DNA is extracted from cell nucleus and broken up into multiple pieces using restriction enzymes

2. Multiple copies of this DNA are produced

3. Double stranded DNA separated into single strands

4. Primers bind to one stand and initiate the replication process

5. The second strand of DNA is recreated by adding complimentary nucleotides using Taq DNA polymerase

6. ddNTP stops the nucleotide sequence

7. The strands are compared to determine the nucleotide sequence

Steps of the sanger method

1. When a dideoxynucleotide that has been marks with the flourescent dye attaches randomly, the new strand terminates

2. By performing 4 separate rections, 4 separate sets of chain-terminated fragments are produced

3. These new strands are various lengths

4. They can be separated using gel electrophoresis, when current is run through the agarose gel and the buffer sits in, the DNA fragments move through the gel from the negative to positive teminal.

5. The larger molecules move slower through the matrix therefore are found near the start/negative terminal, vice versa

6. Markers (which have set fragment sizes) are also run in one lane to compare the unknown fragments to determine their sizes.

Uses of DNA sequencing

Can be used to sequence any segment of DNA.

Can detect mutations and will show whether an individual has a particular disease. Hence, enabling them to seek treatment or prevent the disease.

Used for maternity or paternity tests.

DNA profile definition

Individual people can be identified from samples of human tissue in this way, commonly used in forensics to identify criminals.

Advantages of DNA profiling

• DNA lasting a long time in the environment (dried blood can last 3-4 yrs

• Requires only a very small amount of tissue

• Is individual to each person and can be used to identify relationships

• Tracing ancestry

• Can identify hereditary dieseases

  • probability of genetic disease

  • early detection

non-coding DNA

Consists of many highly repetitive sequences of bases. The number of times the sequences are repeated is subject to mutation and therefore unique to an individual organism.

Species

A group of individuals that can interbreed to produce fertile offspring.

Allele

Different version of the same gene

Gene pool

Sum of all alleles in a population

Population

A group of organisms of the same species living in the same place at the same time.

Mutation

Permanent change in DNA sequecne, due to mistakes when copied or enviornmnetal factors.

Mutagen

Agents known to increase the rate of mutations.

Allele frequency

The frequency of alleles in a gene pool for a particular population

2 types of mutations

• Gene mutations

  • Changes to a single gene during replication where traits are changed or destroyed

• Chromosomal Mutation

  • All or part of a chromosome is affected

Causes of Mutations

• Induced

  • mutations are caused by mutagenic agents in the environment

• Spontaneous

  • caused by random errors in biological processes such as meiosis or mitosis

Somatic mutation

• Body cells

  • mutation passed on to daughter cells during cell division

  • Includes cancerous growth

Germline mutation

• reproductive cells

  • muation passed onto embryo

  • parent doesnt have the mutation

4 effect of the mutation

• missense mutations

• nonsense mutation

• neutral mutations

• silent mutation

Missense muations

Cause a change in the AA and thereore in the protein produced

Nonsense mutations

Change in the base sequence to the cost to STOP. This means that the synthesis of the protein will stop and so a shorter protein is produced that is unlikely to be able to fulfil its function.

Neutral mutations

Cause a change in AA; however, the AA is of the same type and doesn’t change the structure of the protein enough to change its function.

Silent mutations

Doesn’t cause any change in the AA and therefore in the protein produced. This is possible as most AA are coded for by more than one base sequence.

Point mutation

A change in just one nucleotide of DNA

Substiution

Nucleotide replaced with another

Insertion

New nucleotide added to DNA strand

Deletion

Nucleotide removed from DNA strand

Frameshift mutations

Occurs when bases are added or removed. Sometimes results in a different triplet code and AA.

Examples of effects of gene mutations

• Albinism

• Cystic fibrosis

  • Mutation of gene on chromosome 7

  • recessive condition

  • protein which regulates chloride ions across a cell membrane isn’t produced

  • salty skin, persistent cough, wheezing, digestive problems, pneumonia

• Duchenne

  • Wasting of the keg muscles and arms, shoulder and chest

  • Apparent around age 3-5 years.

  • Death occurs due to failure of the respiratory muscles

Lethal recessives

Tay-Sachs Diseases (TSD)

• Recessive condition preventing the production of enzymes

• Accumulation of fatting tissue it the nervous tissue of a new-born baby

• Mental an physical disabilities arise resulting in death

Types of chromosomal mutation

• Deletion

  • chromosome segment lost

• Translocation

  • A segnment from a chromosome is transferred to another

• Duplication

  • A segment from one chromosome is transferred to its homologous chromosome, giving it duplicate genes

• Inversion

  • A segment of a chromosome arm is inverted

Non-disjuction

Where a chromosome pair doesnt separate during meiosis.

Aneuploidy: one daughter cell has one extra chromosome and another daughter cell have one less than the normal number.

E.g. Downs Syndrome (trisomy 21).

Trisomy Examples

• Downs syndrome

• Patau syndrome

• Klinefelter’s syndrome

Down syndrome

• Extra chromosome 21

• Flat face

• Upward slanted eyes

• protruding tongie

• small head

• relatively short fingers

• poor muscle tone

Monosomy

• Cri-du-chat syndrome

• Turners syndrome

Cri-du-chat syndrome

• Missing portion of chromosome 5

• An infant has problems with its nervous system and laryn

• cries like a meowing cat

Identifying Abnormalities

Can be diagnosed before birth through:

• Blood samples

• aminocentesis

• Chorionic villi sampling

How does variation occur

• Mutations (new/different alleles)

• Random fertilisation (infinite combinations alleles in an offspring due to different sperm fertilising different eggs

• Random assortment (random pairing of chromosomes during meiosis

• Chiasmata (exchange of genetic material during meiosis

• Non-disjunction (one or more pairs of chromosome fail to separate during meiosis

Natural selection

Organisms better adapted to their environment tend to survive and pass on the more advantageous traits to their offspring.

Organisms with less desirable traits are less likely to survive and so the less advantageous traits are not passed on.

Over many generations, there is a change in the number and type of alleles in a gene pool.

There needs to be variation in the population.

Struggle for existence, not all the offspring produced would survive to reproduce.

Random genetic drift

Random variation in gene frequency.

Populations of organisms are constantly changing and adapting to their environment. Drastic changes in environmental conditions can sometimes cause drastic changes to the gene pool of the population.

The founder effect

• a type of genetic drift

• loss of genetic variation (small allele frequency) that occurs when a new population is established by a very small number of individuals from a larger population

• E.g. Venezuela Huntingtons disease

An example of the founder effect

• Northwest Venezuela has a high frequency of Huntingtons Disease

• One woman who moved into the area in the 19th century

• She had 10 children who remained in the area

• Now a population of 20,000 people with a high risk of having huntingtons

The bottleneck effect

When some catastrophe like an earthquake or a tsunami kills off most of a population at random and leaves only a handful of survivors.

E.g. Pingelap colour blindness

Example of the Bottleneck effect

• In 1775 a typhoon struck pingelap, only about 20 islanders survived

• From this population and the geographical and cultural isolation, colour blindness increased in the Pingelap population

• Today, up to 5% of the population have colour blindness, and 30% are carriers.

Migration

Effects the number and type of alleles in a gene pool, known as gene flow.

Immigrants bring alleles that are not currently in the population and so the frequency of these alleles will change.

Effects of Barriers to gene flow

• May prevent populations from interbreeding

• this results in different characteristics due to environmental pressures

• Overtime populations differ so much that speciation may occur

• Hence, resulting in the formation of new and distinct species

Geographical barriers to gene flow

• oceans/rivers/lakes

• mountain ranges

• deserts

• ice

Sociocultural barriers to gene flow

• economic status

• educational background

• social positions

How can genetic diseases change a gene pool?

As alleles resulting in fatal disease should be eliminated from a population, although some persist in certaon populations. Hence, changing the allele frequency in a gene pool.

Example of a genetic disease

Sickle-cell anaemia

• Crescent shaped RBCs, reducing O2 carrying capacity

• RBCs can stick together and block blood vessels

• Effects Black Africans, especially in areas where malaria is present

• Heterozygous = resistance to malaria

Evolution definition

Change in heritable traits of biological populations over successive generation.

Variation

Differences between species passed onto offspring. Lead to the idea of ‘survival of the fittest’. Caused by meiosis, fertilisation and mutation

How is competition caused

When food supply and other resources doesnt match rising birth rates.

Survival of the fittest

Organisms with favourable traits survived, those with less favourable traits died before passing on characteristics.

Body structure in Humans

• short-bodied/long limbed human = large SA:VOL

• Long bodied/short limbed human = small SA:VOL

• Neanderthals were more heavily built that modern humans

Speciation definition

Where one species splits into 2 or more separate species so they can no longer interbreed.

How does speciation occur?

• Variation

  • Differences develop within a species over time due to selection pressures

• Isolation

  • groups in a population are physically prevented from interbreeding

• Selection

  • Natural selection causes changes to prevent breeding

DNA sequencing

Determines the sequences of nucleotide bases in a DNA sample. Uses special nucleotides called dideoxynucleotides (ddNTP)

How is gel electrophoresis done?

• DNA samples are loaded into wells at one end of a gel

• The current is run through the gel and the buffer it sits in

• The DNA fragments move through the gel from the negative terminal to the positive terminal

• separate molecules, such as nucleic acids or proteins, based on their size, shape, and electric charge.

Polymerase Chain Reaction (PCR)

Amplify a particular piece of DNA. A laboratory version of DNA replication in cells

Materials needed for PCR

1. DNA sample

2. Taq polymerase

3. Nucleotides

4. Pair of primers

5. Buffer for the correct pH

Why is taq polymerase used?

It was taken form hot springs and hence has a maximal activity at 75-80*.

Due to PCR using very high temperatures a heat-stable polymerase is used otherwise it would denature.

Steps of PCR

1. Denaturation of DNA

   • At 95*C

   • Breaks hydrogen bonds, separating the 2 strands

2. Annealing

   • At 40-65*C

   • Primers bind to complementary sequences on the single strands of DNA

3. Extension

   • At 72*C

   • Taq polymerase extends the DNA chain, adding nucleotides to the 3’ ends of the primers

Uses of PCR

• DNA from a single sperm cell or strand of hair can be amplified for DNA analysis and gel electrophoresis

• fragments of interest in an organisms DNA can be amplified by choosing the right primers

• the selectivity of primers can help identify the likelihood of an individual carrying a particular allele of a gene

Selectivity of primers and how theyre used for disease testing

The longer the primer the more selective it is.

Primers can be created to only bind and amplify certain alleles of genes or mutations of genes