Gene Classification · Web viewThe ABO blood system is also an example of co-dominance as both the IA and IB alleles are expressed in the heterozygote (AB Blood group). However the

AREA OF STUDY 1. – MOLECULAR GENETICS AND HEREDITY

Molecular genetics

The study of the inheritance of traits within a species is known as Heredity. Classical genetics as a study of heredity commenced with the work of Gregor Mendel in the 19th century and deals with the mechanisms and patterns of inheritance through the coded chemical instructions (genes which are the units of inheritance) from one generation to the next. Early studies focused on single genes. Now that we have gained much more knowledge of the biochemical mechanism for the inheritance of traits our attention has turned more to the Genome – the entire set of genes found in the cells of an organism and the matter in which they interact and are expressed.

Chromosomes, Genes and Alleles

This section, repeats a little of what we considered in Unit 3, but it is knowledge critical to understanding what follows in the later sessions this week.

Chromosomes are the structures – made up of the chemical compound deoxyribonucleic acid (DNA) – which carry our genes. In humans there are 46 chromosomes arranged in 23 pairs. Of these 23 pairs, 22 are referred to as autosomal and one pair is referred to as the sex chromosomes. In humans XX is female and XY is male. Diploid (2n) = 46 chromosomes and Haploid (n) = 23 chromosomes.

In eukaryotes, each chromosome is made of two strands of DNA joined in a double helix, each chromosome therefore carries two alleles for each gene, one which has been inherited from the mother the other from the father. These two alleles may be the same, or may be different. A normal individual can only carry two alleles for any single trait at a single gene locus, but in a population there may be many possible alleles which allows for variation within the species. Prokaryotes have only a single strand of DNA and therefore an individual organism can only carry one allele for that trait.

Gene Classification

Genes are sections of a chromosome that contain information for the production of a particular protein. They are classified as either structural, regulatory or homoeotic:Structural genes – produce proteins that become part of the structure and function of the organismRegulatory genes – produce proteins that control the action of other genesHomoeotic genes – produce proteins that play a role in embryonic development of the individual organism

Gene Activity

Genes are switched on or off during our lives. Some are important during childhood and some during adolescence. Also in different cells certain genes will be switched on and others off. For example, genes to make epidermal tissue are of little value for eye tissue cells

Headstart Revision Classes Unit 4 Biology

DNA Structure

The chemical responsible for inheritance of features is called Deoxyribonucleic Acid (DNA). It is comprised of four nucleotides. The nucleotides are made of a sugar group, a phosphate group and a Nitrogen base. Two strands of bases joined by weak Hydrogen bonds between the Nitrogen bases make a double helix chain. The DNA double helix was described by James Watson and Francis Crick in 1953 and earned them the Nobel Prize for Chemistry.

Features of the DNA structure: Basic unit: nucleotide, which comprises a:

o Sugar unit (S)– deoxyribose sugaro Phosphate group (P)o Nitrogen base - one of four chemicals: adenine (A), thymine (T), guanine (G),

and cytosine (C) they pair in the following combinations A – T and C – G.

Complementary base pairs: A only pairs with T; C only pairs with G

Hydrogen bonds between the complementary nitrogen bases hold the two strands togetherA has a double bond to TC has a triple bond to G

Overall the molecule has a negative charge due to the presence of the phosphate group.

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Protein Synthesis

Transcription is the process by which the DNA strands are unzipped, either for the full length of the strand, or just for the segment coding for a protein and copied into mRNA.

1. RNA polymerase attaches to a region of DNA and unwinds the double stranded DNA and exposes the bases of the template strand.

2. The base sequence of the DNA template guides the building of a complementary copy of the mRNA sequence.

3. The result is a single-stranded molecule called pre-mRNA.4. The introns are removed to produce mRNA5. The final mRNA molecule is chemically capped and a poly-A tail added to produce

the operational mRNA.

Before the pre mRNA leaves the nucleus it undergoes a number of modifications to become mRNA. A methylated cap (a modified guanine nucleotide that has a methyl and phosphate group bonded to it) is added to the 5’ end as soon as the mRNA leaves the DNA template.

The Introns are removed by RNA splicing. The introns are regions of base sequences not translated into amino acid sequences (non-coding regions of DNA). Exons are the coding regions that contain the information for protein formation.

Translation

The mRNA passes through the nuclear membrane into the cytoplasm and attaches to sub-microscopic organelles known as ribosomes. Amino acids are brought to the ribosome by tRNA which corresponds to the triplet code on the mRNA.

The Amino Acids are joined together at the ribosome to make a protein.

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Comparison of DNA and RNA

DNA RNASugar unit Deoxyribose OxyriboseNitrogen bases A, T, G, C A, U, G, CDouble/single stranded Double SingleLocation Nucleus Mostly Cytoplasm

The outcome of transcription and translation is a functional protein.

DNA complimentary strand ATG GTC GCC GGC AGA TGA (not copied)DNA template strand TAC CAG CGG CCG TCT ACT (copied)mRNA AUG GUC GCC GGC AGA UGAtRNA UAC CAG CGG CCG UCU ACUamino acid sequence Start, Val, Ala, Gly, Arg, Stop (a very short protein)

Genetic Code

The main features of the genetic code are: Pieces of information in the genetic code consist of triplets or three-base sequences. The code is non-overlapping (that is; bases are read three at a time in discrete triplets). The code is universal (same in all organisms, except a few protozoa and bacteria). The code is said to be redundant or degenerate since, more than one triplet of bases

codes for one particular amino acid. The information encoded in DNA are instructions to assemble amino acid sub-units into

proteins The information is unambiguous (one codon codes for only one amino acid) The information also includes a START instruction and a STOP instruction

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Cell reproduction

The cell cycle is the process by which cells are replaced or more are grown as the normal process of maintenance and repair of the organism. It is a continuous process made up of five stages:1. G1 phase – cell growth prior to DNA replication (Interphase)2. S phase – DNA replication (see below) (Interphase)3. G2 phase – cell prepares for division into two (interphase)4. M phase – mitosis – the nucleus divides (see below)5.C phase – cytokinesis – the division of the cell

DNA Replication (S phase) is the term used to describe the process by which the cell copies its genetic material. All mammals begin life as a single zygote which is the result of a fusion of gametes (sex cells containing only one set of chromosomes), but as adults we are made of trillions of cells. This requires much cell growth. The replication process occurs in 4 steps:1. Two strands of DNA untwist and unwind2. Two strands separate - Hydrogen bonds (which are weak) are broken3. Free nucleotides which are abundant in the cytoplasm come in and pair up with their complementary base. DNA polymerases (enzymes) help the nucleotides become attached to the exposed bases.4. DNA ligase (another enzyme) joins the new stretches of nucleotides into a new strand. The new strand is built in the opposite direction from the original strand. The site where the new strands are being built is known as the replicating fork.

Replication is semi-conservative which means that each new molecule contains one existing parent strand and one new daughter strand

During Mitosis the two chromatids (the two strands of a single chromosome joined together at the centromere) separate. The nuclear membrane breaks down and the chromatids move to opposite poles of the cell. The nuclear membrane reforms and the cytoplasm divides to produce two new cells (C phase). Mitosis occurs in all organisms that are actively growing. During this process one cell divides and produces 2 daughter cells that are identical to the parent cell. These cells have two complete sets of chromosomes are therefore described as diploid or 2n cells.

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Meiosis is a similar process of cell division, however, it produces 4 daughter cells that contain half the genetic information of the parent or one copy of each homologous pair (haploid cells - n). Gametes – sperm and eggs – are made in special organs called gonads – testes and ovaries, by Meiosis. The first Division separates the homologous pairs and the second division pulls the chromatids apart so that the 4 haploid cells may form.

Division I (a normal mitotic division)Interphase I – chromatids uncoilProphase I – DNA replication, potential for single gene mutations, the chromosomes pairMetaphase I – the chromosomes line-up randomly at the equator (this is called independent assortment). During this crossing over may occur.Anaphase I – the chromosomes move to the poles.Telophase I – 2 daughter cells form (diploid)

Homologous pairs of chromosomes are required for meiosis to produce fertile gametes.

Division IIProphase II – chromosomes disentangle and move to equatorMetaphase II – chromosomes line up at equatorAnaphase II – chromosomes move to the polesTelophase II – 4 cells with 23 chromosomes are formed (haploid)

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Comparison of Mitosis and Meiosis

MITOSIS MEIOSISNumber of replications One OneNumber of divisions One TwoNumber of daughter cells produced

Two Four

Chromosome number (haploid /diploid)

Diploid Haploid

Type of cells produced Somatic Cells GametesVariation Minimal Highly Possible

Apoptosis is the programmed death of cells. Cells cannot live forever and each type of cell has a preprogrammed age. Interestingly, cancerous cells are a problem because apoptosis does not occur and they continue to grow and spread throughout the body.

Karyotpes

It is possible to sample a person’s genetic information and make a chromosome map called a karyotype. This can be done by chorionic villus sampling (a fine needle is passed up the vagina and through the cervix to collect tissue in the fluid from around a developing embryo), amniocentesis (a fine needle is passed through the abdominal wall, wall of the uterus and samples fluid from around the developing embryo) and tissue sampling – sperm, mucous, skin, hair, mouth swabs supply cells containing a nucleus which will contain the genetic information of the individual.

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The dotted line represents the position of the centromere.

A homologous pair of chromosomes is a matching pair of same shape and structure. In human males that is 22 pairs of homologous chromosomes and one non-homologous pair (XY) and in human females there are 23 homologous pairs.

In mammals sex is determined by the fathers sperm as he can pass on an X or a Y chromosome, while the mother can only pass on X chromosomes.

X XX X

XXX

Y XY XY

In birds and some reptiles and amphibians the situation is reversed and the mother determines the sex of the offspring.

Z WZ ZZ ZWZ ZZ ZW

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Males are therefore ZZ and females ZW.

Inheritance at one gene locus

Monohybrid Autosomal Crosses

A monohybrid autosomal cross involves alleles of only one gene at a single locus. If the parents’ genotypes are known, the predicted ratio of offspring can be calculated using a Punnett Square.

Mother’s Allele 1 (A) Mother’s Allele 2 (a)

Father’s Allele 1 (A) AA Aa

Father’s Allele 2 (a) Aa aa

A Punnett Square shows the chance of each type of offspring occurring and are often required on your exam (when in doubt include one anyway). In the example above, the two heterozygous parents give a genotypic ratio of 1 homozygous dominant: 2 heterozygous: 1 homozygous recessive or a phenotypic ratio of 3 “Big A’s” : 1 “little a”.

Complete Dominance

In many situations the pattern of inheritance is described as being completely dominant. That is that the phenotypic expression of one allele completely masks the phenotypic expression of the other allele. The former allele is said to be dominant and the latter is described as recessive. These are described with letters of the alphabet. Capital letters are used to denote the dominant allele and the recessive alleles are given a lower case letter, eg:

B – big nosed allele dominant to b – small nosed allele.BB (homozygous dominant individual – would have a big nose)Bb (heterozygous individual – would have a big nose)bb (homozygous recessive individual – would have a small nose)

We can use Punnett squares to describe how complete dominance works. If a homozygous dominant male mates with a homozygous recessive female then:

B Bb Bb Bbb Bb Bb

All offspring have big noses regardless of gender.

If two of the heterozygous individuals are then mated, thenB b

B BB Bbb Bb bb

This gives the genotypic ratio of 1 dominant homozygote: 2 heterozygotes: 1 recessive homozygote. The phenotypic ratio is 3 big nosed individuals: 1 small nosed individual. These are the standard ratios for a heterozygous cross.

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Co-Dominance and Multiple Alleles

For some traits there are more than two alleles. A classic example of this is the ABO blood system. There are three alleles for this trait, but an individual only ever has two of them.

IA – antigen A producedIB – antigen B producedi – no antigen produced

Because there are more than two alleles present greater number of genotypes and hence phenotypes are possible.

The ABO blood system is also an example of co-dominance as both the I A and IB alleles are expressed in the heterozygote (AB Blood group). However the third allele i, is recessive to both IA and IB.

Phenotype GenotypeA Type Blood IA IA , IA iB Type Blood IB IB , IB iAB Type Blood IA IB

O Type Blood i i

Another example is the inheritance of flower colour in peas; ifW for white flowersR for red flowers, then WW – whiteRW – pinkRR – red

We can use Punnett squares to describe how incomplete dominance works. If a homozygous white flower mates with a homozygous red flower then:

W WR RW RWR RW RW

If two of the heterozygous individuals are then mated, thenR W

R RR RWW RW WW

The phenotypic ratio and the genotypic ratio are the same - 1 red homozygote: 2 pink heterozygotes: 1 white homozygote.

Lethal Alleles

Lethal alleles often cause death of the embryo or the offspring prior to reproductive age. Therefore not all alleles have equal chance of survival. In most cases lethal alleles are recessive so that individuals have a low probability of death, however, there are cases when the lethal gene can be dominant. In this case the probability of death for the individual is high.

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Sex Linkage

A special type of monohybrid inheritance is called sex linkage. Genes carried on either of the chromosomes responsible for sex (X or Y) are said to be sex-linked. A trait can be said to be X-linked inheritance involving genes carried on the X chromosome or Y-linked inheritance involving genes carried on the Y chromosome.

One common example in humans is the inheritance of Colour blindness. Colour blindness is a recessive trait linked to the X chromosome and is more common in males than females. If we cross a Colour blind male with a heterozygous normal for colour vision female then we would have the following Punnett Square and outcomes.

Xb YXB XB Xb XB YXb Xb Xb Xb Y

This would result in one normal female and one colour blind female and one normal male and one colour blind male.

Test Crosses

A test cross is used to find out the genotype of an organism. One crosses an unknown individual with an individual known to be homozygous recessive (said to be pure breeding for the recessive trait) and one records the phenotypes from the cross.

For example, you may wish to work out the genotype of a black eyed dog. You may think that brown eyes are recessive to black eyes.So B – black eyes, b – brown eyesYou believed that you owned a true breeding black eyed dog. You would mate your dog with a brown eyed dog known to be true breeding.

If your hypothesis was correct then the first mating would produce all black dogs as shown in the Punnett Square below. This is the test cross.

B Bb Bb Bbb Bb Bb

To prove this you would then need to breed two of the puppies produced. This is not correctly described as a test cross, but is used to confirm the test cross.

B bB BB Bbb Bb bb



This would give you the phenotypic ratio of 3 black to 1 brown and would be considered as evidence for your theory.

Pedigree Analysis

Pedigrees are used to show the inheritance of a trait or disease in a family and are often used in combination with Punnett Squares to investigate how a certain trait has been passed on or could be passed on.

EXAM TIPSThere are particular modes of inheritance that can easily be observed when looking at a pedigree. One usually needs three generations of a pedigree to be able to make reasonable inferences from the data.

Autosomal recessive inheritance- for an individual to express an autosomal recessive trait both copies of the allele must

present- if both parents affected all offspring will also be affected- recessive traits tend to skip generations and few individuals are affected

Autosomal dominant inheritance- for an autosomal dominant trait to be expressed only one copy of an allele is required- usually present in each generation, many affected individuals

X-linked recessive inheritance- males only require one allele to express the trait, therefore more males than females

will show the trait



- females require both alleles, often females don’t show the trait but act as carriers passing to a majority of their male offspring

X-linked dominant inheritance- any individual with the trait must have a parent with the trait- females may be heterozygous and show the trait- much harder to detect

Inheritance involving two gene loci

Independent Assortment

In the case of the inheritance of two genes on two separate chromosomes (independent assortment) the phenotypes from the two heterozygous individuals are in the ratio 9 dominant for both genes: 3 dominant for one gene: 3 dominant for the other gene: 1 recessive for both genes.

You can demonstrate this with a Punnet square. Two traits A and B with the possible alleles A and a and B and b. Both parents are heterozygous for both traits, that is AaBb. The possible gametes are AB, Ab, aB, ab.

AB Ab aB abAB

AABBAABb AaBB AaBb

Ab AABb AabbAaBb

Aabb

aB AaBBAaBb

aaBB aaBb

Ab AaBb Aabb aaBb aabb

If one of the parents is homozygous for both traits and the other is heterozygous, then the Punnett Square offers a different outcome.

AB Ab aB abAB

AABBAABb AaBB AaBb

All the offspring will look the same displaying both the dominant phenotypes, although they all have different genotypes.

Variation

Variation is the degree of difference that exists between members of a population. The greater the degree of variation, the more genetically healthy is a population, ie more able to respond to change in the environment.

If there is only one gene with two alleles for a particular trait, you either express the trait or you don’t. Example: the allele for tongue rolling is dominant to the allele for not being able to roll



your tongue. That means within a population there is very little variation, you either can roll your tongue or you can’t. This is known as discrete variation.

If there is more than one gene for a particular trait and if each gene has multiple alleles, such as those that control height, or skin colour, there is much greater variation within the population and it usually follows a normal distribution pattern. This is known as continuous variation.

The Sources of Variation are: Multiple Alleles and Poly Genes – as discussed above the more alleles present for a

particular gene and the more genes involved in a certain trait, then the greater the source of variation.

Crossing Over in Meiosis – during meiosis it is possible for chromatids to become entangle and to break and reform differently causing variation by rearranging the normal groups of alleles present in the gametes

Environment – has an influence over the phenotypic expression of the genotype, thus increasing variation within a species, eg. Diet and nutrition affect the size of individuals.

Independent Assortment – normal fertilization sees the alleles being assorted independently – thus sexual reproduction assists with the increase in variation.

Random Fertilisation – plants are more able to hybridise by mixing gametes between like species and so further variation occurs.

Mutations

Mutations

Mutation – is a change in the DNA sequence (this can have a variety of effects)

During DNA replication or when a cell is undergoing mitosis and meiosis, errors can occasionally occur which result in a mutation. Mutations occur naturally. However the mutation rate can be increased by factors which cause instability in DNA. These include:

high temperatures certain chemicals known as mutagens, such as asbestos and many chemicals in cigarette

smoke radiation – particularly high energy forms such as those in X rays and in nuclear reactors.

Mutations can occur in: germline cells – this means that they can be passed to the next generation somatic cells – these mutations are confined to the organism the mutation occurs in and

are therefore not passed on.

Mutations cause:

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ContinuousVariation

DiscreteVariation

number of individual

number of individuals

14


new alleles in the population – so a source of further variation new phenotypes which can be acted on by natural selection

Types of mutations: Mutations can be changes in just one single base (gene mutations) OR they can be changes where thousands of bases are involved (chromosome mutations)

There are four types of gene mutations. Point Mutations – are those gene mutations that occur at a single base Deletion of a base – the removal of a complete base Addition of a base – the addition of a complete base

Deletion and addition mutations cause frameshift mutations (when the reading frame is changed). Some frameshift mutations involve a stop codon being produced - this is known as a nonsense mutation.

The substitution of a base. This mutation only affects one codon, and a different amino acid may be incorporated into the protein. If so, the phenotype could be severely affected. This type of mutation is known as a missense mutation.

Some substitution mutations produce no change to the protein being produced. If AGA, which codes for serine, is altered by substitution to AGG then the amino acid serine will still be coded for. This is known as a silent mutation. For example, AGA is transcribed to UCU, and AGG to UCC.

Inversion mutations. This is when two or more bases are reversed; it may produce a slightly different functional protein. eg, GAG becomes AGG and causes a change from leucine to serine.

Template DNA TAC TTC ACG GCA ATC CGA

Deletion TAC TCA CGG CAA TCC GA?

Addition TTA CTT CAC GGC AAT CCG A??

Substitution TAT TTC ACG GCA ATC CGA

Inversion TAC TTC AGC GCA ATC CGA

Chromosomal Mutations

There are four types of chromosomal mutations. These mutations usually affect much more of the chromosome than gene mutations and so their effect is usually much greater.

Deletion – a region of the chromosome is deleted all together Duplication – a region of the chromosome is duplicated Inversion – a region of the chromosome is turned around Translocation – a region of a DNA from one chromosome is added to another

chromosome



All of these changes, whether to large sections of the chromosome, or to single bases can change the protein being coded for so that it no longer works at all, works – but does something else, or it may have no effect at all.

Genotype and Phenotype

The Genotype of an individual is the alleles an individual carries for a particular gene. For example: Alleles for eye colour (autosomal inheritance) can be denoted as B – brown or b – blue. Therefore a Homozygous individual carries two identical alleles for a particular trait eg BB, bb. Heterozygous individuals carry two different alleles for a particular trait eg Bb

The Phenotype of an individual is the visible characteristics an organism displays for a particular trait, for example eye colour, the ability to roll your tongue or the presence or absence of a disease. These arise from their genotype as well as the influences of the environment.

Environmental influences on Phenotype

phenotype = genetics + environment

Some characteristics are predominantly determined by genetics, such as the ability to roll you tongue, you either can or can’t do it. Non tongue rollers will never be able to roll their tongue, no matter how much they practice.

Other characteristics are determined by both your environment and your genetics. You may have fair skin, but when exposed to sunlight it changes colour to a light brown colour. You may possess the alleles for tallness inherited from one or both parents, but because of poor nutrition or disease during a critical stage of your development, you may not achieve the potential height that you alleles code for.

Gene Technology and its Applications

REVERSE TRANSCRIPTASE

This is an enzyme used to make copy DNA (single stranded DNA) from messenger RNA. Often mRNA can be obtained, which contains no introns, only the coding regions of a gene. This is particularly useful for inserting genes into other organisms as many organisms will only accept small amounts of DNA.

A Poly – A tail is added to the mRNA, this then acts as the region for the primer to bind to. Reverse transcriptase, a naturally occurring enzyme, catalyses the addition of new bases to the cDNA. Once this is made, the mRNA is removed and DNA polymerase is added to build the complementary strand of DNA. The result is double stranded DNA created from the mRNA base.

These enzymes were discovered in viruses. The enzyme is encoded and used by reverse-transcribing viruses, which use the enzyme during the process of replication. Reverse-transcribing RNA viruses, such as retroviruses, use the enzyme to reverse-transcribe their RNA genomes into DNA, which is then integrated into the host genome and replicated along with it.


http://en.wikipedia.org/wiki/Genome

http://en.wikipedia.org/wiki/Retrovirus

http://en.wikipedia.org/wiki/RNA_virus

http://en.wikipedia.org/wiki/Reverse-transcribing_virus

http://en.wikipedia.org/wiki/Reverse-transcribing_virus


HIV infects humans with the use of this enzyme. Without reverse transcriptase, the viral genome would not be able to incorporate into the host cell, resulting in the failure of the ability to replicate. Understanding and then replicating this process has allowed medical scientists to find treatments, cures and vaccines for retroviruses.

ENDONUCLEASES (RESTRICTION ENZYMES)

Restriction enzymes are used to cut pieces of DNA. They have specific recognition sequences where they cut. They can produce either sticky ends or blunt ends. They can be used to insert pieces of foreign DNA into bacteria or virus. The bacterial DNA and the gene to be inserted in the bacteria are cut with the same restriction enzyme, to ensure their ends match.

Blunt ends Sticky ends

Restriction enzymes are naturally occurring proteins often found in bacteria. They are named after the bacteria and numbered in order of their discovery. For example:EcoRI – the first restriction enzyme isolated from the bacteria Escherichia coliAluI – the first restriction enzyme isolated from the bacteria Arthrobacter luteus

RECOMBINING DNA

DNA ligase is used to join fragments of DNA together. Once DNA has been cut by a restriction enzyme and the new piece of DNA is ready to insert, DNA ligase is used to join the nucleotides together. DNA ligase occurs naturally in all organisms and is used to zip up the DNA strands after they have been copied in natural protein synthesis.

VECTORS

Vectors are commonly used to transport DNA from one organism to anotherThere are two main types of vectors: plasmids and viruses.

Plasmids:These are small circular parts of DNA that have come from bacteria. Genes can be inserted into these plasmids (using restriction enzymes and DNA ligase) and transported to another organism or put back into the bacteria, once it has been modified. Plasmids are commonly used by genetic engineers. For example, Insulin is now produced in vast quantities using bacteria. The human gene for the production of insulin is inserted into a plasmid. The bacteria are then grown with the insulin gene and caused to produce insulin which is harvested and sold to insulin diabetics. In the past insulin was taken from the pancreas of dead pigs, and sometimes sheep and cattle. It was expensive, some people had allergic reactions to it and others had religious, moral or ethical objections to using the animal insulin.



Plasmids which have been modified (genetically engineered) are easily taken up by bacteria. These cells are then said to have been transformed.

Viruses:Viruses by nature infect other cells and insert their DNA or RNA in the process. Genetic engineers can insert foreign DNA into viruses and target cells. An attempt was made to use viruses as a vector to treat Cystic Fibrosis. Individuals with this disease produce a very thick mucous in their respiratory system (as well as thick mucous at other mucous membranes and other symptoms) due to a point mutation. The viruses were transformed to carry the correct gene coding for the protein that makes the mucous and inhaled by the sufferer. A number of problems occurred with this gene therapy and it was withdrawn from sale.

POLYMERASE CHAIN REACTION (PCR)

PCR is a method used to amplify DNA (make many copies of it). Often the sample off body tissue you have only contains a small amount of DNA. PCR is used to make many millions of copies quickly. PCR is an artificial way of DNA replication. PCR is a simple 3-stage process that is repeated 25 – 30 times.

Stage 1 Denaturation. The DNA is heated to 95oC and the strands separate as the H bonds are brokenStage 2 Attach primers. The solution is cooled to 50oC and primers (nucleotide bases) bind to both strands of the DNA.Stage 3 Extension. Raise the temperature to 72oC the new strands are completed.

Repeat as often as required.



GENE PROBES

A gene probe is a small piece of single stranded DNA or RNA that has been radioactively labelled or labelled with a flourescent marker. Gene probes are used to locate target sequences of DNA; that is, a known nucleotide sequence that is unique to the gene in question. They tend to be 20 – 40 nucleotides in length, but can be up to 1000 and are complementary for a known sequence. The sequence must be complementary to the target sequence to be able to find the correct piece of DNA in the sample being studied. This is commonly used when testing an individual for the presence of the gene responsible for an inherited disease.



ELECTROPHORESIS

This is a method for separating DNA fragments according to size. The DNA sample is put into wells in a gel. The gel floats in a buffer solution and a current is passed through the solution. The DNA fragments, being slightly negative in charge moves toward the positive end of the gel. Smaller fragments move faster through the gel than larger ones, with fragments of the same size being aligned as shown in the diagram below.

DNA PROFILES

There are regions in DNA which vary in length in individuals, as they contain repeated sequences of DNA eg GAGAGAGA. These regions are inherited, just like alleles, thus individuals are unique. The size of the repeated sequences can be shown on an electrophoresis gel and this is known as a DNA Profile. There are two types:STR – short tandem repeats – section of non-coding DNA of between 2 and 5 bases. Eg. the dinucleotide GA is repeated many times to form the STR. These are inherited and an individual has a distinctive number based upon heredity.VNTR – variable nucleotide tandem repeats – also a section of non-coding DNA, but they are longer than 5 bases.



DNA SEQUENCING

Using Gel Electrophoresis it is possible to establish the order of each nucleotide in a strand of DNA (that is the sequence of the nucleotides). This can be used to map the location of genes as in a species based Genome project or to study a particular mutation or disorder.

Manual Sequencing – this is often called the Sanger method and uses gel electrophoresis. The gel is left to run for 1 – 3 hours. Small samples are sequenced by a trained technician analyzing the location of the different nucleotides.

Automatic Sequencing – when a large amount of DNA needs to be sequenced this can be done reasonably quickly by machine. The four nucleotides are fluorescently labeled and a laser scanner reads the single lane of a gel sending the information to a computer which reconstructs the base sequence. These machines can be left to run for 10 hours at a time.

MOLECULAR CLONING

Cloning means to make an identical copy. By using these techniques it has become possible for scientists to remove sequences of nucleotides which are known to code for a particular protein, insert them in another organism and have the gene function to produce a particular effect

An example of this, described above, is the addition of the human insulin gene to a bacterial plasmid so that the bacteria can be used to produce insulin for use by diabetics. To achieve this scientists needed to sequence the gene for human insulin, find restriction enzymes that would cut the DNA in the appropriate places, find a gene probe that could be used to remove the insulin gene and then transform the bacterial plasmid.



APPLICATIONS

MEDICINE

Treatment of Cancer: Most Cancers are caused by sporadic mutations of somatic cells and can not be passed on, but some are regularly seen in families suggesting a change in the gametes for that family. Gene technologies can be used to assess the possibilities of particular cancers occurring, assessing the mutation that has caused a cancer and applying this information to the treatment of the cancer. Medical scientists are working on gene therapies to treat cancers, such as injecting genes for toxic proteins into the cancerous cells.Pharmaceuticals: The production of drugs (like insulin as discussed earlier) using transformations of bacterial plasmids with genes from other organisms which code for a particular drug.Gene Therapy: Some diseases are caused by the lack of a functioning gene. If the gene can be isolated, inserted into a vector (such as a virus) and then the vector can be used to introduce a functioning copy of the gene into the affected cells. This was attempted with Cystic Fibrosis as discussed earlier.Stem Cell research: The discovery of totipotent embryonic stem cells (cells that can be caused to become any other cell) and omnipotent adult stem cells (cells that can still be caused to become a lot of other types of cells) has raised the possibility of using a patient’s own cells to grow specific tissue. For example, injecting stem cells into damaged regions of the brain and growing new nerve tissue or growing skin tissue to be used as a graft.

MALNUTRITIONMalnutrition is caused by both the lack of food and the lack of food with sufficient nutrition value. Many crop species, including rice, wheat and sorghum – common food staples in many cultures, are being transformed to become richer sources of nutrients such as Vitamin A. The vitamin enriched crops are then grown by the community to ensure that they receive both food and food of a reasonable nutritional quality.

AGRICULTUREMany agricultural crop species are being transformed to confer protection against diseases such as cotton, canola, corn and wheat. These transformed crops often have a genetic resistance to pesticides introduced so that the farmer can apply pesticides more efficiently and not worry about the effect on his crop.

CONSERVATIONFor a species to be viable, it requires genetic diversity. Researchers are using the DNA profiles of many endangered species to better manage the existing populations. They can use this information to ensure individuals with different profiles are mated and that individuals with very similar profiles are not mated. Also, they are able to identify populations that are distinct enough to be considered either sub-species or alternative species. The decision then has to be made whether to combine the genetic information and reverse the evolutionary process or to keep the populations distinct.

LEGAL USES



As you are all no doubt aware from watching television, these techniques can be used in the solution of crimes. People leave their DNA behind them wherever they go. The presence of a person’s DNA at a crime scene indicates that they were present at the location. The police still need to prove that the person was there at the time the crime took place and were involved in that crime. DNA profiling also allows cases of paternity to be solved.

SUGGESTED ACTIVITIES

1. Arrange 20 concepts or ideas from this part of the course and list them in order of know well to know least well.

2. Prepare flash cards for each of these 20 concepts/ideas. Idea on one side and explanation on the other.

3. Select 20 concepts or ideas from this section of the course and create a mix and match activity. That is, you have a list of 20 concepts or ideas and a list of 20 definitions that have been mismatched. You then share your list with someone else and try to solve each other’s lists.

4. Write your own quiz. Prepare 20 multiple choice or True/False questions for a friend. Have your friend prepare 20 questions for you. Try each other’s questions.

5. Take 5 of these processes and create a poster for each that explains the process and its potential use. Remember a poster is a graphical representation of the information – more drawings than text.

6. Choose the 5 you are least comfortable with – you might like to list the processes and put them in rank order from know well to know least well first.


Documents

Gene Classification · Web viewThe ABO blood system is also an example of co-dominance as both the IA and IB alleles are expressed in the heterozygote (AB Blood group). However the