Genetic Biology Mc Stay Lectures 1-5

Fundametals of chromosomes

Each human cell comprises of 2 genomes:…

gene encoding regions?

organisations?

Nuclear genome and Mitochondrial genome

Nuclear - only small part encodes genes

-coding elements are spread out - complex, flexible genetics

Mitochondrial genome

-Almost ENTIRELY gene encoding

Highly compact - simple minimalistic

Human karyotype

The complete set of chromosomes in a human cell

shows size,shape structure

1-22 are autosomes

23 - sex chromosomes

-females = 2 x chromosomes

-Males = (x,y)

Karyotypes - used to study genetic abnormalities

such as DOWN SYNDROME - extra copy of chromosome 21 (triosomy)

Mitosis

cell division

-Results in 2 genetically identical daughter cells

-each have same number of chromosomes as parent cell

-Mitosis

-Ensures BOTH daughter cells have an IDENTICAL GENOME to that of the parent cell - barring errors

Crucial for growth, development and tissue repair

Phases of the cell cycle are….

Explain the phases E

Cell cycle

-Series of events leading to cell DIVISION AND REPLICATION

replication first - makes the sister chromatids - THEN MITOSIS

M phase: Cell division phase - Mitosis and Cytokinesis -CHROMOSOMES ARE CONDENSED

Stages in mitosis: Prophase,prometaphase metaphase,anaphase,telophase,cytokinesis )

Chromosomes condense in preparation for nuclear and cell division

After replication - each chromosome consists of 2 SISTER CHROMATIDS joined by centomere - bound by cohensins

Chromatids aligned along metaphase plate - PULLED APART DURING ANAPHASE- cohesins cleaved

Pulled to opposite poles

Cytokinesis - Cytoplasm divides - forming 2 daughter cells - each with a full set of chromosomes

(SISTER CHROMATIDS SPLIT FORMING 2 DAUGHTER CELLS)

Interphase: Growth + DNA replication - CHROMOSOMES ARE DECONDENSED

G1 - period of cell growth, cell ensures it has the nutrients required to grow

S PHASE:

Dna replication phase - replication of Chromosomes - form - held together by cohesins to be cleaved during anaphase

G2 - further growth and preparation for MITOSIS

Explain the structure of the chromosomes and the behaviour with each phase

Before s phase

Chromsomes contain ONE double helix

-Relaxed + decondensed

After s phase

Sister chromatids - each containing a DNA DUPLEX (double helix)

Each chromatid - contains a single dna double helix

Chromatids joined tightly at the centromere with COHESINS

-allow them to function as a single chromsome

During m phase

-Chromsomes condense

-Align at metaplate

-Cleaved cohesins during anaphase to separate the sister chromatids into individual chromsomes with IDENTICAL genomes

-EQUAL DISTRIBUTION of independent chromosomes after anaphase.

After replication what occurs>

Replication - sister chromatids made

Mitosis now

-Separation of the sister chromatids - held together by cohesin complexes

-Chromosomes condense (shorten + thicken)

Centrioles migrate to opposite poles+ form spindle poles

Prometaphase - NUCLEAR MEMBRANE breaks down - allows for anaphase to occur but also for chromosomes to align along the metaphase plate

Metaphase - residual cohesins removed

-Chromosomes aligned along metaphase plate - cohesins and centromere hold sister chromatid together.

Anaphase - spindles pull sister chromatids apart to opposite poles

Telophase:

reformation of the nuclear membrane around daughter nuclei

-Chromosomes decondense

cytokinesis - separation of the cytoplasm

creates 2 daughter cells

G1 - S PHASE -G2 -M PHASE

ACTIN + MYOSIN CONTRACTILE RING PINCHES CELL

Sexual reproduction

-Genetic mutation was slow prior to this

sex - adaptive feature - common to almost ALL multicellular organisms

-Extremely efficient mechanism for PRODUCING VARIATION

Allowing organisms to ADAPT to changing environments.

Meiosis is key

-Cell division - iN SEX CELLS

What is meiosis in terms of sexual reproduction:

How many rounds of division

Variation?

Cell division - sexually reproducing organisms use it to produce GAMETES - sperm and egg cells.

2 round of division occur

Meiosis I and Meiosis II

Ultimately result in 4 CELLS with one copy of each paternal and maternal chromosome

2 forms of genetic variation introduced

-Recombination - between homologous chromosomes

-Independent Assortment of chromosomes

Meosis - how many rounds of cell division

Before or after replication

-how many chromosomes?

What happens before meosis

What is S - phase referred to as?

Meiosis - Specialised reproductive cell division

-During meosis - genetic combinations are randomly created

-Exchanging sequences between maternal and paternal chromosomes BUT ALSO by independent assortment.

AFTER replications - produces GAMETES

2 rounds - 4 DAUGHTER cells - ½ the chromosomes of the parent cell (gametes fuse to get full no of chromosomes)

Before meiosis - replication

2 identical sister chromatids - held together by sister chromatid cohesion

s phase = Premeiotic S - phase

Monopolin homologs - ensure regulated separation of sister chromatids

What happens IMMEDIATELY AFTER DNA replication?

over view

Think G1 - S -G2 -M

After s = G2 phase

MEOSIS - Sex cell division - formation of gametes

Meiotic prophase - (prolonged G2 stage)

GENETIC RECOMBINATION

Homologous chromosomes PAIR + UNDERGO genetic recombination

(One chromsomes from the mother and one from the father pair up) - Synapsis

During spermatogenesis X and Y chromosomes CANNOT PAIR ALONG THEIR LENGTH

Pairing only occurs at pseudo-autosomal regions at the end of each chromosome

Recombination process involves

Cutting + Repairing of DNA - allowing exchange of genetic information. - specialised enzymes create double strand breaks in the DNA of the chromatids of homologous chromosomes.

After the breaks - DNA is repaired by exchange of chromatids between homologous chromosomes - Results in the MIXING OF GENETIC MATERIAL between the mother and the father - creating

Recombination:
Part of one of the maternal chromosome is swapped with part of the paternal homologous chromosome

Cross overs:

Segments of chromatids from homologous chromosomes are exchanged

Chromatids exchange genetic material through chiasmata - Physical links in homologous chromosomes where genetic material is exchanged

RECAP: GENETIC diversity - meiosis - genetic recombination

Meiosis - Specialised reproductive cell division

-During meosis - genetic combinations are randomly created

-Exchanging sequences between maternal and paternal chromosomes BUT ALSO by independent assortment.

Prophase I

Meosis - sex cell division

STAGE 1

How is recombination initiated?

LEPTOCENE

-Formation of synaptomenal complex - condensing of chromsomes

-Double strand break initiation by SPO11

Longest phase of meiosis

Chromatid exchange

-Maternal and paternal chromsomes pair, synapse + exchange genetic information - BY GENETIC RECOMBINATION (break + repair)

-Form at least ONE crossover per chromosome

Stage 1

-Leptotene stage

Chromosomes condense - become visible under a microscope

-Sister chromatids held together by cohesin

Chromosome individualisation occurs - Individual chromosomes become distinct visible threads

Chromosomes arranged as array of loops - loops extend from the AXIAL ELEMENT

Condensing of chromosomes - forms synaptonemal complexes

-helps tight pairing of chromosomes

Lateral elements of the synaptonemal complex form along each chromosome - forming the axial element - from this loops of the chromosomes extend - each containing a portion of chromosomal DNA

Synaptomenal complex - crucial for correct alignment of chromosomes for recombination

Chromosome organisation

Allows chromosomes to find and pair with their homologous partner in ZYGOTENE

LEPTOTENE - aids recobmination - exchange of genetic information leading to genetic diversity

DOUBLE STRAND BREAK INITIATION (needed for genetic recombination to occur)

Enzyme SPO11 creating programmed double strand breaks

Step 2 in prophase meiosis

Zygotene

-CHROMOSOME PAIRING - BIVALENT FORMATION

Synaptomenal complex is formed

-Homologous chromsomes are much more closely and stably paired - Due to the formation of the synaptomenal complex

-2 chromosomes paired together - form bivalent structure - stabilised by s.c.

prepared for genetic recombination

Recombination is already beginning with pairing stabilised by s.c

Prophase step 3

Pachytene

(patching up the breaks)

completion of crossing over + genetic recombination

Repair of double strand breaks

-Homologous recombination - including chromosomal cross over

-Completion - by REPAIR of DOUBLE STRANDED BREAKS created in leptotene

Most repaired WITHOUT FORMING CROSS OVERS - At least one cross over per chromosome

Subset of breaks cause cross over -

-Cross over / exchange of genetic material between non sister chromosomes (non homologous)

Causes genetic diversity in offspring

prophase step 4

Diplotene

SLIGHT SEPARATION - still connected in some parts (chiasmata) - chromosomes become more DI

-Synaptomenal complex disassembles - homologous chromosomes SEPARATE A LITTLE

Homologous chromosomes of each bivalent

-Remain tightly bound to chiasmata - the regions where crossing - over occured

Chiasmata - ensure correct chromosome segregation - without them - nondisjunction could occur ( improper chromosome segregation)

Chiasmata remain on the chromosomes until they are SEVERED at the transition to anaphase I - to allow the homologous chromosomes to separate + move to opposite poles of the cell

Prophase steps

L,Z,P,D. - THINK lspd but z

Anaphase of meiosis

2 PARTS

1 AND 2

ANAPHASE - MEIOSIS ONE

• HOMOLOGOUS CHROMOSOME SEPARATION

-homologous chromosomes - segregate away from each other

-2 HAPLOID CELLS ½ number of chromosomes of the parent cell

gre

ANAPHASE - MEIOSIS TWO

Sister chromatids segregate - COHESION BETWEEN SISTER CHROMATIDS IS RELEASED

Segregate from each other

4 haploid cells

Meiosis 2 differs to mitosis because

chromsomes have mix of genetic information due to recombination in prophase

MEOSIS GENETIC VARIATION

-Genetic recombination between homologous chromosomes

(as discussed)

-prophase - leptocene, zygotene, pacytene, diplotene

Break and repair - cross over and recombination

segregation - meiosis 1 and 2 = homologous chromosomes separate 2 = cohesion removed sister chromatids separate 4 cells ½ number of chromosomes of parent

REMEMBER GAMETES HAVE ½ NUMBER OF CHROMOSOMES

OTHER MODE OF VARIATION - Independent assortment

independent assortment

The 23 chromosomes are aligned and separated in such a way that the combination of MATERNAL AND PATERNAL chromosomes in the resulting gametes is random

SPERM AND EGG CELLS HAVE A MIX OF CHROMOSOMES

Homologous chromosomes are RANDOMLY DISTRIBUTED to different daughter cells during anaphase 1 of meiosis

(ONE FROM MOM AND ONE FROM DAD)

23 CHROMOSOMES

2²³ COMBINATIONS possible

-High genetic diversity

Structure of chromosome recap

The dna inside the chromosome is compacted into a chromatin structure

dna in the chromatin structure is wrapped around octamer of core histones

-forms a nucleosome

Heterochromatin: - dna is tightly bound to the histone - hard access - inactive transcription

Euchromatin - more relaxed dna - easier to access - active transcription

Interphase - chromosomes are less condensed - NOT tightly condensed

Metaphase - chromosomes are TIGHTLY condensed

mitosis and meiosis - condensing of chromosomes - allows for accurate division - individualisation

autosomes - non sex chromosomes 22 of them

sex chromosomes 1 pair (X,X) OR (X,Y) female / male

Specialised regions in human chromosomes - what are the functions?

What are most humans

Specialised regions

-Ensure complete replication + accurate segregation during cell division

-centromeres and telomeres

-Most humans are metacentric - centromere near the middle

-5 chromosomes = acrocentric - short p-arm - contains little / no genetic information

submetacentric - slightly off center

Play a role in the genetic function of the chromosome

Down syndrome = trisomy of chromosome 21

Centromeres

function?

-TYPES / ranges of centromeres

POINT / REGIONAL

SMALL + DEFINED / LARGE + REPETITIVE - RELY ON KINETOCHORES

centromere - region of DNA on the chromosomes - acts as POINT of attachment for the kinetochore

-Crucial during cell division

ensure correct segregation of chromosomes into daughter cells

ranges point - regional -

Point centromeres:
Yeast - least complex

-very short, small, well defined dna

-Specific - dont involve large repetitive DNA

Centromere sequence - essential for attachment of spindle to microtubules + correct chromosome segregation

Regional centromeres:
Higher eukaryotes

-More complex

Large + repetitive regions of sequences of DNA

Long arrays of repetitive dna sequences - satellite dna repeat arrays

Satellite dna

-Non coding

-essential for centromere function + proper chromosome segregation

Alpha satellite dna

-Long tandem repeats

-Necessary for centromere formation

repetitive nature stabilise the centromere + enables the correct attachment of kinetochore proteins involved in chromosome movement

Evolution and Centromere Identity:

• In higher eukaryotes, centromeres are not defined by a single, simple DNA sequence but rather by specific chromatin modifications and the presence of kinetochore proteins that allow for correct chromosome segregation.

• Centromere identity is not solely determined by the DNA sequence but is also influenced by epigenetic factors, including modifications like histone variants and DNA methylation. This is why centromeres can be shifted or redefined in certain species or conditions.

• Yeast (e.g., Saccharomyces cerevisiae): Yeast centromeres are composed of a short, defined sequence (point centromeres) that is crucial for accurate chromosome segregation.

• Humans and other mammals: Centromeres consist of long arrays of repetitive DNA sequences, such as α-satellite DNA, and rely heavily on kinetochore proteins for function.

centromeres - Formation of the kinetochore

a histone protein H3 is replaced by a VARIANT HISTONE CENP-A

-stabilises the structure

-Forms on the centromere

-Recruits kinetochore proteins to form the kinetochore

CENP-A acts as a SCAFFOLD for the formation of the kinetochore protein

Telomeres

function

How is this carried out?

-Protect the ENDS of linear chromosomes

-Cap the chromosomes - PREVENT INTERNAL DNA being degraded by nucleases

-solve the end replication problem

• Maintained and extended by telomerase enzyme

• Telomeres can shorten with cell division - without telomerase this would cause cell death.- counteracts shortening

telomerase - uses reverse transcriptase + noncoding RNA template to make new TELOMERE dna repeates

-TERT - telomerase reverse transcriptase is an RNA-dependent DNA polymerase - synthesis of DNA from an RNA template

uses RNA template provided by its other subunit TERC - telomerase RNA component.

(terc) The rna template contains a sequence that is complementary to the telomere repeat sequence in humans - allowing tert to use terc to add more dna repeats to the telomere

complementary sequence = hexanucleotide sequence in the rna template

TERT USES TERC to add telomere repeat sequences to chromosome ends

Telomeres

Shelterin complex

telomere = the protective cap on the ends of chromosomes - consists of the shelterin complex - prevents internal dna from being degraded by nucleases

Specialised set of proteins - protects chromosomes ends- prevents them from being interpreted as dna double strand breaks

(genomic instability would ensue)

Telomeres:

TRF1 AND TRF2 - proteins that bind to double-stranded telomeric repeats - responsbile for

TRF2 - lengthening the telomere

TRF1 - protection + forming the protective t- loop that prevents recognition as a double strand break

POT1 - interacts with the single-strand 3’ overhang of the telomere dna - TTAGGG

Binding proteins

TIN2 AND TPP1 -connect POT1 to trf1 + trf2 - help complete the shelterin complex

Rap 1 - binds to trf2 - contributing to stability + protection of the telomere

prevents inappropriate activation of DNA damage response

t-loop creates lariat like structure - physically protecting the chromosome ends

-t -loop shields chromosome ends -prevents them from being recognised as a double strand break

Maintain genomic stability, prevent aging + cancer

importance of centromere and telomeres

-Faithfully transmit DNA from mother to daughter cells

centromere - correct segregation at cell division

telomere - cap the chromosomes - prevents the internal dna from being degraded by nucleases

-prevent inappropriate dna damage response

Lecture 2

Patterns of Inheritance and how to study and identify chromosome

Definitions

Locus

Alleles

Genotype

Phenotypes

Locus - the unique chromosomal location - position of the gene on the chromosome

-defines the position of an individual gene or DNA sequence

ABO blood locus group (example)

Alleles - alternative form of the same gene - a,b,o

genotype - List of the alleles present at one / several loci

phenotype - observable traits / character(istics) of an organisms

-simple inspection to sophisticated lab investigations (range)

more terms:

zygote

homozygous

heterozygous

autosomal / x linked

dominant

recessive

Zygote - union of the sperm cell and the egg cell

Homozygous - if both alleles at the locus are the same

heterozygous - both alleles at the locus are different

Autosomal / x linked character

-depending on the location of the gene on the chromosome

-few y linked characters

autosomal character - on the non sex genes

-not sex-dependent

-can be dominant / recessive

x-linked (not many y linked)

-females can be carriers if they have 1 x chromosome mutation

-More chance for mutation to occur as they have 2 X chromosomes

Males are (X,Y)

-males are most affected - have 1 chromosome (only 1 no back up like females)

Dominant - if heterozygous and CHARACTER OBSERVED

Recessive - if heterozygous and character NOT OBSERVED

Gene

-A functional unit of dna

-a determinant / co- determinant of a character that is inherited

gene encodes one protein - have sequences that encode the protein included in the dna

HUMAN GENES - UPPERCASE ITALICIZED NAMES

Monogenic vs multifactorial inheritance

(genotype = list of alleles at locus)

Monogenic

-Genetic characters whose presence / absence depends on the GENOTYPE AT A SINGLE LOCUS

-Mendelian characters - pedigree patterns

Non-mendelian / MULTIFACTORIAL

-GENOTYPE AT MULTIPLE LOCI

-Genetic characters are governed by genes at MORE THAN ONE locus

-governed by a small number of loci = oligogenic

-governed by lots of loci, each with small effects = polygenic

Mendelian characters are usually described as…

-Described as dichotomous - you have it or you don’t e.g. extra finger

Cystic Fibrosis

HUMAN TRAITS

-Non - mendelian - not dichotomous

Most a CONTINUOUS / QUANTITATIVE CHARACTERS e.g. height / weight

-We all have them, just to different degrees

Continuous traits - QTLs - quantitative trait locis

mendelian pedigree patterns

square - man

circle = woman

dot = carrier

blue = affected

single line = mating

autosomal dominant inheritance

NOT AFFECTED BY SEX (XX OR XY)

50% chance

• an Affected person will usually have an affected parent

  TRANSMITS by either sex

  AUTO SOMAL DOMINANT INHERITANCE

Child one affected / one unaffected parent

-50% chance of being affected

½ chance of being affected = risk of being affected = 50%

Autosomal recessive inheritance

Affected people usually born to unaffected parents

-Mum and Dad are carriers (blue dot)

-parents usually asymptomatic carriers

-affects either sex

-AFTER AFFECTED CHILD BORN

-Each child has 25% chance of being affected

X- linked recessive condition

Recessive - parents usaully not affected

-Affects mainly males

-Males usually affected have unaffected parents

-MOTHER USUALLY A CARRIER (asymptomatic)
Has affected male relatives

Can females be affected?
Yes - if father is affected + mother is a carrier

individual marked with ?

Would be 1 in 2 if male (MOTHER PASSES X, FATHER PASSES Y) MOTHER HAS 2 X so ½ chance they get her faulty chromsome

In recessive - female must inherit 2 FAULTY COPIES - one from mother and one from father (because females have 2 x chromosomes)

Carrier mothers have a 50% chance of passing the affected X chromosome to their sons, meaning the risk for an affected male child is 1 in 2 (50%).

x-linked dominant

1 parent IS AFFECTED

-Affects either sex but MORE FEMALES THAN MALES

-Females often more variably affected - due to having 2 x chromosomes

child of affected female - 50% chance of being affected

affected male - all his daughter but no son affected

(son not affected bc dad has disease and he passes y chromosome not the faulty x chromosome to his son)

Risk that individual marked with a query would be affect = 100% if female

0% if male (male passes y chromosome to son)

MALES PASS THEIR X CHROMOSOME TO THEIR DAUGHTERS

BUT ONLY THEIR Y CHROMOSOME TO THEIR SONS -

if father affected - the x chromosome passed to the daughter is affected

recap

recessive - usually parent not affected

recessive - daughter needs to get faulty gene from both parents

x-linked

Father GIVES HIS Y CHROMOSOME TO HIS SON

If father affected - boy will not be affected, girl WILL be affected

FATHER PASSES HIS ONLY X chromosome to his daughter

-the condition is DOMINANT which means only 1 is needed

mother (x,x)

father (x,y) - y to son, x to daughter (y linked diseases extremely rare)

Complications to basic patterns

Why?

name an: autosomal dominant + autosomal recessive disorder

Variable expression of individuals within the same family due to genetic background, modifier influences, environmental factors

Human characters showing dominant character can skip a generation due to variation in genetic background - nutrition, exercise and medication can lessen the severity (sometime)

sometimes can inherit a mild and aggressive version of the mutation in the same gene - can partially restore the function

VARIABLE GENETIC BACKGROUNDS

Waardenburg - autosomal dominant trait

Cystic fibrosis = autosomal recessive trait

Different family members show different features of type 1 waardenburg syndrome - an autosomal dominant trait

-although they have the same mutation in the PAX3 gene that encodes a transcription factor.

A UNIQUE GENETIC background can surpress the mutant allele (e.g. gene silencing) but the offspring can still inherit the mutant

Complications to the basic patterns

-Discussed - genetic background causing variation in the expression of the faulty caused by the same mutation (waardenburg syndrome)- hearing loss, premature greying, white forelock, different coloured eyes

would expect 100% penetrance but…

sometimes due to random variation

extreme variable expression

• dominant condition may fail to manifest itself

• “skipping a generation”

• variable faulty gene expression - due to genetic background influenaces

Age - related penetrance

Disease example?

-Huntington disease - neurodegenerative disorder

severity and probability of the symptoms depends on the age of an individual

Huntington proteins expressed in neurons in the brain - uncontrolled expansion - leads to the death of neurons and large gaps in the brain

A = probability of an individual carrying the disease allele developing symptoms by a given age

B = asymptomatic person who has an AFFECTED parent carries the disease allele

• should undergo genetic testing to check if they carry the hd allele (HD allele will be present from birth)

• even if an individual shows no symptoms, they may emerge with age.

  neurodegeneration with age

Huntington gene protein expressed in neurons in the brain- glutamine in gene encoded by codon cagu - stretch of glutamine -

• Copying of gene and replication and repair - CAGU can expand - above 36 copies - uncontrollable expanding Huntington protein - large stretches of glutamine

• Protein precipitates - kill neurons - large gaps in the brain - removed neurons

Happens with age

Huntingtons disease = autosomal dominant inheritance (dominant usually has an affected parent)

50% chance of passing it down to each child ( not sex related)

MOSAICISM AND NEW MUTATIONS

Mosaicism - Individual has 2 or more genetically different cell lines, derived from one original zygote

Post zygotic genetic change, meaning the mutations occurs AFTER fertilisation

Occurs

-Single cell is mutated after fertilisation

-Mutated cell DIVIDES -forms a population of cells genetically different from the rest of the cells in the body

straight AFTER FERTILISATION - mutated cells will contribute to a large number of tissues in the body -

If happens later not immediately after -mutated cells will contribute to a much lower number of tissues in the body (tissue - restricted mosacism) milder, localised manifestation)

IF MUTATION occurs EARLY IN DEVELOPMENT (AFTER fertilisation) - this affects a large number of individuals cells + contributes to a large number of tissues in the body..

• The person may show signs of the mutation (phenotypic signs)

Later in development

• Tissue-restricted - may not have the phenotype, or may have milder symptoms

Mosaic downsyndrome

-later stage mutation

some cells have the extra chromosome, some don’t - milder symptoms in the individual than if they had an extra chromosome in each cell

Another example is mosaic Huntington's disease, where the mutation in the HTT gene occurs later in life, and only certain tissues may show symptoms of the disease, leading to a milder or later onset of the disease.

CHIMERISM

chimeras

Fusion of 2 zygotes to form a single embryo - form 2 genetically different cell lines

2 early stage embryos fuse during development

Often can have no visible impact on an individual

Chimerism - rare + proved by presence of too many parental alleles at several loci in a sample that is prepared from a large number of cells

Too many alleles in the same tissue on different loci - usually an individual inherits one allele from each parent

if testing a chimera - multiple tissues could show more than 2 alleles

New mutations are often present in … form?

what does this mean

COMPLICATING PEDIGREE INTERPRETATION

NEW MUTATIONS

(Genetic variation due to genetic background) another one

Parent may produce a single mutant gamete

post-zygotic expression - chimeras / mosaicsm

Often present in mosiac form

-This means they werent inherited from parents

-Mutation occured during the development of the embryo - long after or shortly after fertilisation

transmitted mutation may have occured

-Mutation in the germline ( cells that contribute to sperm / eggs) - it can be passed to offspring

mutation in non germline cells - wont be passed to offspring

PROBLEMS WITH SIMPLE PEDIGREE representation

1. Variation in genetic expression

2. age-related penetrance

3. new mutations

studying human chromosomes

methods:

Array Comparative genomic hybridisation (CGH)

Whole genome sequencing will replace all these methods

Karyotyping under the microscope

preparing a metaphase spread

-What is an important step?

Preparing metaphase spreads

-Disrupt the mitotic spindle using a microtubule poison such as colchicine

-Pauses the cell in metaphase

prevents aligning along the metaphase plate to progress to anaphase

-Colchicine inhibits microtubule polymerization - binds to tubulin blocking mitosis

Preparation of metaphase spreads

Karyotyping

how are chromosomes identified?

metaphase - easiest to analyse for karyotyping

-We want to create a full set of chromosomes

how?

Using a blood sample - extract 5ml of venous blood (want white blood cells, specifically lymphocytes - easily cultured + have a nucleus

1. extract 5ml venous blood sample

2. Add phytohemagglutinin (induces mitosis for clear karyotyping) and Sample is placed in a culture medium at 37 degrees celcius for 3 days

3. Add colchicine + harvest the cells and re-suspend in hypotonic saline - water enters the cells by osmosis - cells swell and are easy to visualise for chromosome analysis

4. fix cells in methano / acetic acid - preserves the chromosomes

5. digest with trypsin and stain with GIEMSA

   -trypsin digests proteins on the chromosome surface such a histones - make the DNA easier to stain

   Giemsa - binds to dna - creates a distinct pattern of light and dark bands along the chromosomes

   Trypsin - controlled digestion

Identifying chromosomes

-identified by SIZE + BANDING PATTERN

Positively staining bands are known as G-BANDS (think g for Giemsa)

How are banding patterns displayed?

-banding seen depends on?
How are chromosomes grouped??

What is a satellite?

As chromosomal ideograms

-Banding seen depends on microscope resolution

different banding resolutions to resolve bands

Grouping

-Autosomes are numbered from largest to smallest BUT

chromosomes 21 - slightly smaller than chromosome 22

-Metacentric - centronmere is near the middle

Submetacentric - centromere at or near the end (think submarine under water)

Satelite - small segement separated by NONCENTROMERIC CONSTRICTION from the rest of a chromosome

-These occur on the SHORT ARMS of most acrocentric human chromosomes

Fundamentals of chromosomes mitosis + meiosis (genetic recombo)

Patterns + inheritance - identifying and studying chromosomes (metaphase spread) protection for genetic transfer

Chromsomal abnormalities - lecture 3

LECTURE 3

Chromosomal abnormalities

2 types

1. Polyploidy and aneuploidy

2. Structural chromosome abnormalities

Polyploidy

2 types

Triploidy - 3 full sets of chromosomes -fertilisation error

triploid embryo

1-3% of human pregnancies

-triploid embryo

-Rarely survive to term - not compatible with life (embryos)

error in fertilisation

Dispermy - single egg fertilised by 2 sperm - accounts for 66% of triploidy

Diploid ovum - diploid egg fertilised by normal sperm 10%

Diploid sperm 24% - Diploid sperm fertilises normal egg

-Diploid = incorrect meiosis occured

Tetraploidy - 4 full sets of chromosomes - ENDOMITOSIS cell division error

Normal fertilisation + fusion of gametes

AFTER FERTILISATION -incorrect cell division

NORMAL ZYGOTE

-Endomitosis - DNA REPLICATES without subsequent cell division

dna replicates BUT CELLS FAIL TO DIVIDE

Leads to ONE LARGE CELL WITH TWICE THE Normal amount of DNA

Lethal - result in miscarriage

Rare mosaic form - some tetraploid some normal diploid cells

-Survival still rare

Aneuploidy vs Euploidy

What is the cause of Aneuploidy?

Euploidy = having complete chromosome sets

polyploidy = having multiple chromosome sets

Aneuploidy - one or more individual chromosomes are present as an extra copy or are missing

-Abnormal number of chromosomes that is not a multiple of the haploid number

Aneuploidy - cause = nondisjunction / anaphase lag

(errors in chromsome separation during meosis / mitosis

How do aneuploid cells arise

Explain

-Usually through nondisjunction in maternal meiosis

Failure of homologous chromosomes to separate properly during cell division

3 FORMS OF NONDISJUNCTION

(instead of separation of chromosomes - each go to the same cell and become fertilised)

1. Failure of a pair of homologous chromosomes to separate in meosis

2. failure of sister chromatids to separate during meosis II

3. Failure of sister chromatids to separate during mitosis

Nondisjunction during meiosis

produces gametes with 22 / 24 chromosomes

-Reasons not very clear - most cases due to maternal meiosis

Nondisjunction

produces one MONOSOMIC cell and one TRISOMIC daughter cell

Monosomic cell usually dies

-Trisomic cell can survive and establish MOSAIC triosomy

-Nondisjunction

-Anaphase lag

Aneuploidy

ANEUPLOIDY

-misalignment / lagging

Misalignment

Sister chromatids FAIL to align with the metaphase plate

-Remain at one of the spindle poles causing aneuploidy

-BOTH chromatids can enter ONE DAUGHTER CELL

trisomy / monosomy

Anaphase lag

-When a chromosome / chromatid is DELAYED in its movement during anaphase

-Lags behind the others + fails to be incorporated into the daughter nucleus

-Chromosomes that do not enter the nucleus of a daughter cell are eventually DEGRADED

or SOMETIMES persist in micronuclei as small membrane - bound vesicles

ANAPHASE LAG MECHANISM

-involves Merotelic attachements

Instead of the kinetochore being attached to one spindle pole, it is attached to microtubules from both spindle poles

-PUSH ME PULL ME EFFECT

-the chromosomes are pushed + pulled but dont move

lagging - may be excluded from the daughter cell

Cliical consequences of Aneuploidy

-TRISOMY

• some survive to term? others dont? exception?

-MONOSOMY

-Usually lethal

-Multiple abnormalities present at birth (congenital)

-Trisomy - extra chromosome - even though the chromosome is perfectly normal - congenital implications (present at birth)

-Trisomy 13 and 18 - CAN SURVIVE TO TERM -sever malformations - incompatible with long term survival

Most autosomal trisomies - NOT COMPATIBLE WITH SURVIVAL

unless mosaic form (only some cells are affected, while others have the normal amount of chromosomes) - early development stages

MONOSOMY

Autosomal MONOSOMIES - EVEN MORE CONSEQUENCES - Lethal, catastrophic

ANEUPLOIDY - Imbalance of different chromosomes and genes e.g. transcription factors

monsomies - MORE PROFOUND EFFECT

-one missing worse than having 3

DOWN SYNDROME IS A TRISOMY OF CHROMOSOME 21

EXTRA SEX CHROMOSOME (STRAYING FROM AUTOSOMES

In females - random inactivation

• FAR FEWER ill effects

• -Can function normally or have relatively minor problems compared to AUTOSOMAL TRISOMY - 47 xxx 47 XXY - can function normally

Monosomy in sex chromosomes - fewer consequences

45,X - turner syndrome

turner syndrome

45 Y is lethal - y chromosome is essential for male development BUT does not contain the genes required for survival (located in the x chromosome)

-short, infertile, learning disabilities, hearing loss etc.

Random inactivation of X CHROMOSOME IN FEMALES

In females, (XX) one of the X chromosomes is randomly inactivated - One x chromosome remains active per cell

Ensures, like men, women have a similar x-linked expression (men have 1 x chromosome and ONE Y)

-TURNER SYNDROME

One X chromosome is missing - the single x remains active in cells - no x inactivation occurs.

x- chromosomes - contain ESSENTIAL GENES

AUTOSOMAL CLINICAL COSEQUENCES ARE WORSE THAN SEX CHROMOSOMES

How do CHROMOSOME STRUCTURAL VARIANTS EMERGE?

-Mis repair of damage -especially DNA DOUBLE STRAND BREAKS, can be recombination errors / replication errors.

Shelterin - telomerase protection the ends of chromosomes - prevents them from being recognised as a double strand break - prevent INAPPROPRIATE DNA damage response. (deletions, duplications, inversions) inappropriate recombination - during meiosis

Without telomeres - ends of chromosomes are TREATED as double strand breaks

telomeres - crucial protection + stability of the chromosome

Reciprocal TRANSLOCATIONS

-Structural variants of Chromosomes

EXTENT OF CONSEQUENCE OF RECIPROCAL TRANSLOCATIONS

DEPENDS ON?

• Mis repair of double strand breaks

• -inappropriate recombination (during meiosis) (remember this is where recombination occurs) - obviously recombo problems cause variation

• Errors in replication

Problem

-More than 2 BROKEN ENDS - repair machinery JOINS THEM TO THE WRONG PARTNERS.

2 nonhomologous chromosomes swap segments.

-CHROMOSOMES ARE A BLEND OF THE ORIGINAL CHROMOSOMES

-if each chromosome variant still has ONLY ONE CENTROMERE - then MITOSIS CAN OCCUR WITH NO PROBLEMS

DICENTRIC CHROMOSOME

-Occurs when 2 parts of a chromosomes swap and both RETAIN THEIR CENTROMERE.

Chromosome now has 2 centromeres

-mitotic spindle responsible for chromosome separation - struggles to pull chromosome to opposite poles of the cell

Mitotic spindle CANT DEAL with a chromosome with 2 centromeres.

Breakage - fusion - bridge cycles - MITOSIS OF DICENTRIC CHROMOSOME

-Tension, spindle will try to pull chromosome in two directions at once

Breakage - double stranded break of chromosome

Fusion - non homologous during Reciprocal Translocation

Bridging - during cell division

breakage - separation

Break fusion break - cycle occurs where the segments of the chromosome continuously

Separation of chromosomes fails to successfully occur - ANEUPLOIDY

Cause fragmentation + genomic instability

-HALL MARK OF CANCER IN CELLS - CHROMOSOMAL ABNORMALITY

ACENTRIC CHROMOSOME - ANEUPLOIDY

Chromosome has LOST its centromere due to translocation

-Chromosome CANT be pulled to either pole during mitosis

ACENTRIC CHROMOSOME

-Usually lost during mitosis - fails to segregate into daughter cells due to MISSING CENTROMERE

-Results in genetic loss + instability

ANEUPLOIDY

MITOSIS -

Acentric - loss of genetic material

Dicentric - bridge - fusion - break cycle - MITOTIC SPINDLE CANT HANDLE 2 CENTROMERES - fragmentation of chromosomes

genetic INSTABILITY

Reciprocal Translocation scenarios

-Dependent on?

Another> think breaks? multiple breaks (2)

LOSS OF TELOMERES>

INVERSION = 2 BREAKS

Para = same arm

Peri = different arms

centromere

Dicentric - chromosome has 2 centromeres - (Joining of 2 segments) - mitosis - chromosome strain - bridge -fusion - break

Acentric - chromosome LOST its centromere - lost during meiosis- unable to separate in sister chromatids

2 BREAKS on one chromosome

-Incorrect repair of break + NO LOSS OF MATERIAL

-Paracentric / pericentric inversions

Paracentric - inverted segments DOES NOT contain the centromere

-Broken segment FLIPS but stays within the same arm of the chromosome

-No change in genetic information, BUT THE ORDER OF THE GENES IS CHANGED

-Can cause problems during mitosis - IF CROSS OVERS occur - dicentric / acentric chromosomes may arris

Pericentric - inverted segment contains the centromere

Inversion on different arms - centromere included

Gene order is altered, no genetic material is lost

Crossing over leads to DUPLICATIONS AND DELETIONS IN CHROMOSOMES

-misalignment during meiosis

Unbalanced chromosomal rearrangements in offspring

Congenital abnormalities

-GAMETES fail to fertilise

INVERSION RESULTS IN 2 BREAKS

Paracentric - breaks are on the same arm

Pericentric - breaks are on different arms.

Structural variants

Mis-repair of breakages

-Centromeres - acentric / dicentric - para / peri (arms / centromeres included)

Loss of telomeres - formation of RING CHROMOSOME

-Ends of chromosome fuse together

-Loss by EXTREME SHORTENING

Associated with intellectual disabilities, growth delays + congenital effects

Multiple breaks on a single chromosome

-No loss of material?

names

INVERSIONS

Breakage then inversion

Paracentric - breaks are on the same arm

Then inversion occurs

-Pericentric -breaks are on different arms

Inversion then occurs

The order of the gene is altered- due to inversion

BUT no genetic loss

Loss of telomere / no telomere

ends fuse together - create a ring chromosome

(same arm breakages ) - can produce acentric chromosomes

Repair can produce an acentric fragment - LOST DURING NEXT MITOSIS (missing chromosome) - genetic instability

Interstitial deletion / ring chromosome - fusion of chromosome breakages in a circular manner - absence of telomeres

interstitial genetic loss - segments between the BREAKAGES unable to reattach

ribosomal gene arrays are found on?

-Function of ribosome gene arrays

RNA ONLY FOUND ON P ARMS

p arms - short + consist of repetitive DNA

Only found on P ARMS of acrocentric chromosomes

-CENTROMERE is located AT THE END

Function:

Protein synthesis

Robertsonian translocations

-Joins 2 acrocentric chromosomes - 13,14,15,21,22

-MOST COMMON FORM OF HUMAN TRANSLOCATION

acro-centric chromosomes

-Very small

Largely repetitive ribosomal RNA genes rRNA genes

SAME sequence found in SIMILAR BUT NOT IDENTICAL CHROMOSOMES (ACROCENTRIC)

-RECOMBINATION between homologous sequences in HETEROLOGOUS short arms (not identical but evolutionarily similar origin)

=Acentric - missing centromere

Dicentric products - 2 centromeres one from each chromosome fused together

USUALLY

Two centromeres of the dicentric chromosome are SUFFICIENTLY close together - function as a single large centromere

-1 CENTROMERE IS EPIGENTICALLY SILENCED

-FUSION chromosome SEGREGATES REGULARALY + STABLE IN MITOSIS

-prevents genomic instability + PREVENTS break- fusion - bridge cycles

Acentric fragment- LOST

genetic information is lost - doesnt affect PHENOTYPE

as rDNA repeats are found in high numbers in all other acrocentric chromosomes

• PRESENT IN EVERY 1 IN 1,000 BIRTHS

Consequences of structural chromosome abnormalities

are robertsonian considered balanced?

CAN BALANCED chromosome abnormalities still affect the phenotype?

-give an example of this

abnormalities

Structural variants - TRANSLOCATION (Reciprocal/Robertsonian

-BALANCED?
-No net gain / loss of chromosomal material

balanced - no phenotypic effect (usually)

Unbalanced

-May have an effect

Robertsonian- balanced - no phenotypic affect

-Considered balanced - no phenotypic effect

epigentically silenced centromere

lost centromere - acentric genetic info lost but rDNA is plentiful in other acrocentric chromosomes - loss of genetic material is negligible / redundant

SOME balanced abnormalities AFFECT THE PHENOTYPE

-impercise repair - deletion, duplication, mutation of IMPORTANT GENES

-chromosome break could disrupts an IMPORTANT GENE

-An active gene could be translocated to HETEROCHROMATIN where it is silenced

-Gene is separated from its enhancer

DYSTOPHAN - muscular dystrophy

-Breakage near the DMD gene causes the DMD gene to translocate to heterochromatin- silences the gene causing muscular dystrophy

Over expression? causes?

-Balanced abnormalities - malignant phenotype

C-MYC gene

over expression causes CANCER - GENE AMPLIFICATION

C-MYC GENE - CAUSES CANCER

-Over expression

-Translocation between chromosomes 8 and 14

C-myc gene is translocated/ juxtaposition to immunoglobulin HEAVY CHAIN LOCUS

-MYC overexpression

MCY is upregulated in B lymphocytes

-Malignant phenotype

Uncontrolled Cell proliferation

consequences of structural abnormalities in MEIOSIS

ABNORMALITIES WITH 1 CENTROMERE- balanced

-Transmission through mitosis NO PROBLEMS

-No excess / missing material

Abnormality = Balanced

Normal PHENOTYPE - usually

Exceptions - Muscular dystrophy + BURKITT’S lymphoma

Mitosis is fine

PROBLEMS ARISE IN MEIOSIS THOUGH - Homologous chromosome pairing

-Rearranged (variant) chromosomes DO NOT match their normal counter parts

-Homologous sequences between the chromosomes still pair

Unbalanced gametes

-excess / missing genetic material

Translocations - form CROSS STRUCTURES

Contortions of chromosomes

-BALANCED abnormalities

-No problem getting through mitosis

-Meiosis - cross structures / contorted chromosome structures as HOMOLOGOUS SEQUENCES join together but chromosomes as a whole are different due to variation (improper repairing - double strand breaks - cross over)

• can be a carrier of translocation

• can be normal

• trisomy / monosomy - after fertilisation aneuploidy - maternal meiosis

Remember aneuploidy - disjunction / anaphase lag

-NON DISJUNCTION - leads to mono / trisomy

(remember polyploidy = additional chromosome sets)

consequences of structural chromosomal abnormalities in meiosis

(remember meoisis is where the abnormality is detected BECAUSE chromosomes are not homologous to fuse together - so only fuse partially

-CAUSING ANEUPLOIDY - trisomy / monosomy

ROBERTSONIAN 14:21 - TRANSLOCATION

Robertsonian = acrocentric chromosomes

5% of downsyndrome - due to ROBERTSONIAN TRANSLOCATION

aneuploidy - chromosome 14 is translocated to chromosome 21

trisomy

structural variants

-can be small duplication or deletions

can be large scale dna rearrangements either

Recurrent structural variants

NAHR

-non allelic homologous recombination (alternative forms of the same gene = allele)

OR UNEQUAL CROSS-OVER

-Occurs between 2 repeated sequences

-repeated sequences

-Meiosis - unequal cross over - deletion vs duplication

cross over - non homologous chromosomes EXCHANGE genetic information

unequal cross - over

-1 chromosome - recieves duplications of a region of the genome (duplication)

-1 chromosome loses the region of the genome (deletion)

MISALIGNMENT OF LOW COPY REPEATS

Unequal cross - over - NAHR - NON ALLELIC HOMOLOGOUS RECOMBINATION

LOW COPY REPEATS - REPETITIVE BUT PRESENT IN LOW NUMBERS) - SHARE SIMILAR SEQUENCE

NAHR - SAME ORIENTATION - DUPLICATIONS + DELETIONS

duplication of ABC region Chromosome 1

deletion of ABC region Chromosome 2.

NAHR - DIFFERENT ORIENTATION of the sequence LOW sequence NAREPEATS

reversed orientation of repeats

-can affect gene function

affects gene expression -phenotypic changes can result

NAHR - LOW COPY NUMBER REPEATS

syndrome? recurrent - happens repeatedly

NAHR - Recombination - fusion of non homologous chromosomes WITH (at) homologous sequences - unequal cross - over - duplications / deletions of regions of dna

Prader Willi syndrome - Angelman syndrome

-Duplications / deletions of low copy number repeats due to NAHR

4mB region on 15q11-q13

Phenotype depends on origin of the parent

-In the sperm or the egg?

Maternal = Prader willi - intellectual disabilities, developmental delay

Paternal = Angelman syndrome

non recurrent structural variants

recurrent = NAHR (prader willi / angel man)

non recurrent

Double strand break misrepair

DOUBLE STRAND BREAKS caused by ionising radiation / recombination

-most challenging to repair

repair of DSB

-recombination - like process

Homology directed repair e.g. CRISPR

ANOTHER WAY NHEJ - MORE ERROR PRONE

Non homologous end joining

-Broken ends are stripped back and polished - BROKEN ENDS ARE PROCESSED - (FRAYED ends must be polished)

-single strand overhangs removed

HALLMARK = presence of small deletions AT THE BREAKPOINT - (sometimes insertions)

(DSB FIXED BY NHEJ - CAUSE OF STRUCTURAL VARIATIANTS) NON RECURRENT (recurrent = NAHR insertions / deletions)

small scale deletions / insertions - Small scale variation / mutation

NAHR (recurrent) - larger scale variation than NHEJ (non-recurrent)

how else can double stranded breaks occur

-Replication fork encounters single strand break / dna lesion

-Must repair the break to PREVENT loss of genetic information

-Replication fork stalled by single strand break - leading STRAND IS STALLED

-Lagging strand continues - FORMS single DOUBLE STRAND BREAK - when replication fork collapses

-NHEJ of a double stranded broken end fused to the single DOUBLE strand break

NHEJ - is ERROR PRONE

• can join to the wrong chromosome - chromosomal translocation

• joined in the wrong orientation - inversions of genetic data

• insertions/ deletions at the repair site

insertion,deletion,translocation,inversion (wrong orientation)

translocation - Burkitt’s Lymphoma

what if there is no DOUBLE strand available? for NHEJ?

-No second dsb available? - micro homology - mediated break - induced replication

3’ end of broken strand may INVADE any sequence where there is MICRO HOMOLOGY - 2-6 MATCHING BASE PAIRS

OFTEN invades DNA sequence at another replication fork with a single strand

Replication starts using this sequence

Replication process is stabilised after several cycles

-rearranged chromosome with duplications, deletions and insertions of different segments from various regions.

recurrent vs non recurrent

recurrent - NAHR

-ANGELMAN + PRADER WILI

NONRECURRENT - NHEJ (dsb) + Micro homology-mediated break induced replication (no 2nd dsb)

Nonreciprocal

-Only the sequence is copied from template to patch up dsb on the affected dna

Gene conversion can occur - template strand unaffected - no genetic transfer / reciprocal transfer)

transfer is uneven - all to the broken none to the template

21B inactivated by 21A copied sequence - adrenal hyperplasia

CAH - congenital adrenal hyperplasia

Non-recurrent structural variants

Strand invasion - leading to gene conversion

Double strand break initiates normal recombination

gene conversion = non reciprocal genetic exchange

sequence from one chromosome is copied ONTO THE OTHER WITHOUT EQUAL EXCHANGE

How does it happen?

DSB occurs

-Microhomology induced recombination - 3’ end of broken dna chromosome invades a sequence of DNA with 2-6 matching base pairs

INSTEAD OF CROSS OVER RECOMBINATION

The short sequence is copied from one chromosome to another

No reciprocal transfer occurs

Original template remains unchanged

-(Invaded chromosome is unchanged, sequence is just copied

-Broken chromosome is patched with the copied sequence

21A pseudo gene into 21B

-21A pseudo gene sequence copied onto 21B

-Inactivating mutations

Makes 21B gene nonfunctional - CODING sequence is interrupted

21 hydroxylase enzyme is IMPAIRED

defective cortisol production

Excessive androgen production - adrenal hyperplasia

Conversion of the 21B gene - to inactive form

summary of lecture 3

unequal cross over in NAHR - micro deletions / duplications

DONT AFFECT THE CARRIER

-BALANCED

-Balanced abnormalities

problems during MEIOSIS

Monosomy / trisomy - aneuploidy - improper segregation

e.g. Robertsonian translocation - fine mitosis but not meiosis - trivalent structure

Segregation is improper

monosomy / trisomy due to aneuploidy - nondisjunction / anaphase lag - merotellic attachements

-Miscarriage / congenital syndrome e.g. downsyndrome

How do we find specific DNA sequences in complex mixtures?

DNA - one strand can be used to find the complementary strand

-DNA have such distinct structures - can easily be observed in complex mixtures

dna probes- detect dna

rna probes - study mrna

Nucleic Acid hybridisation

-Well characterised nucleic acid - DNA / oligonucleotide probes used to identify related sequences / complementary sequences in a complex sample

Based on Annealing

2 complementary strands of DNA will bind together to form a double stranded DNA molecule

EXAMPLES

FISH - fluorescence insitu hybridisation

fish - chromosomal abnormalities

FISH - fluorescent dna probes bind to complementary sequences - gel electrophoresis sep fragments - hybridise with dna probe + detect

-Probes detect specific chromosome - 1 signal = monosomy / 3 = trisomy - abnormalities of chromosomes

PCR primer annealing

southern blotting + northern blotting - dna digestion with restriction enzymes

Colony hybridisation

Comparative genome hybridization -test DNA + template DNA - fluorescent labelling check for deletions + duplications in the genome

RED / Green sequence lost of gained?

Denaturing and annealing dna

How is dna denatured

gentle?

Annealing?

Denaturing - double strand breaking of h.b

-Break up the hydrogen bonds in a duplex nucleic acid (double stranded)

• by HEATING / exposure to highly polar chemicals e.g. urea + formamide

• Separated strands can re-anneal to form the original double-stranded dna

  Instead of boiling structure to denature - loss of genetic information

• Instead use urea / formamide to chemically breakdown the bonds

  -can use a physiologically gentle temperature - no damage of genetic information

What affects denaturing and annealing

-The extent of hydrogen bonding (denaturing is breaking up these bonds)

-Temperature - temperature can denature

-Chemical environment - highly polar can denature

Melting temperature 50% of dna dissociates into single strands due to thermal denaturation

Tm = 4°C x (number of G’s and C’s in the primer) +2°C x (number of A’s and t’S IN THE primer)

G and C have MORE HYDROGEN BONDS) G = greater

g and c = 3 hydrogen bonds

a and t = 2 hydrogen bonds

-More hydrogen bonds = higher melting temperature

Salt NACL -stabilises the hydrogen bonds - harder to denature

Strongly polar chemicals e.g. urea + formaldehyde DISRUPT the hydrogen bonds

19mer oligo with 10 gc base pairs

4×10 + 2×9 = 58 degrees

Formation of artificial duplexes + natural homoduplexes

2 DNA SOURCES - duplex = double strands

Denatured

single strands of dna ARE MIXED - homologous sequences

Homoduplex - original strands reanneal after denaturation

heteroduplex - homologous sequences on different strands re-anneal after denaturation

DNA single strands ARE MIXED UP - partially complementary sequences

heteroduplex formation in Nucleic acid hybridization assay

-DNA probe chosen - single defined nucleic acid labelled

All double strands are DENATURED

Single stranded probe nucleic acids are MIXED with single - stranded test sample nucleic acids

Strands with complementary sequences anneal

Reannealing of denatured strands - homoduplex formed

template homoduplex

probe homoduplex

ALSO

Annealing of partially complementary probe and TEST-SAMPLE SEQUENCES - HETERODUPLEX -within complex nucleic acid population

What do most hybridisation assays use?

Denatured dna samples on SOLID SUPPORTS - unlabelled

Denatured + exposed to aqueous solution of denatured labelled nucleic acid probe

Partially complementary sequences of labelled dna probe will bind to sample dna sequences

Other labelled sequences not bound - bound at incorrect locations

-CAN BE WASHED OFF

-Bound nucleic acids can be visualised in this way

Southern Blot hybridization

Blotting - requires restriction endonuclease (enzyme)

-Digest the nucleic acid

-Fragments are applied to agarose gel + separated by size using gel electrophoresis

-Gel treated with ALKALI - DENATURE THE DNA FRAGMENTS.

Placed against a nitrocellulose / nylon membrane

mEMBRANE IS SOAKED in a solution containing a radiolabelled single stranded dna probe

-After hybridization probe is washed - remove excess probe

-Membrane placed against x-ray film

Position of the labelled probe will causes a LATENT IMAGE - hybridization band

Exon sequence capture

magnet?

Exome

Exome = the expressed genome

BIOTINYLATED PROBE + STREPTAVIDIN COATED MAGNET

Genomic dna from patient is fragmented

Biotinylated fragment complementary to sequence of interest is prepared (biotin helps with selective purification later)

Both the fragmented dna + the biotinylated probe are denatured

Reannealing occurs - heteroduplexes form between biotinylated probe sequence + complementary sequence from the patient

Streptavidin-coated magnetic beads added

-heteroduplexes with the biotin group are bound to the beads and selectively removed using a magnet (streptavidin-coated magnet beads)

Elution of the bound patients dna to the probe

-By heating the sample - disrupts the biotin streptavidin-coated beads binding

1. Denature biotin dna probe+ patient dna

2. annealing

3. annealed heteroduplex retrieved by streptavidin (coated) magnetic beads

4. magnet removed by heating (biotinylated probe ALSO REMOVED) patients dna that has the sequence of interest remains

Random primer labelling of Nucleic acid

template dna to be used as the probe is denatured

Single strand of DNA

random primers are added - bind randomly to complementary sequences on the dna template

-dNTP mix is added AND A LABELLED NUCLEOTIDE - usally radioactively labelled

-Nucleotide is entered in region complementary to template

DNA polymerase extension

-Extends the primers

incorporates dNTPS and the labelled nucleotide

-NOW have radioactive DNA probe.

indirect labelling - method of choice

-Dna template of interest with nucleotides carrying biotin - DNA LABELLED WITH BIOTIN

-This probe is allowed to denature + bind to the sample complementary dna - sequence forming a HETERODUPLEX

-After hybridization

streptavidin coated beads - affinity molecule binds to biotin

To detect the probe - visualize the hybridized dna probe - USING REPORTERS

REPORTER = BIOTIN

AFFINITY MOLECULE = STREPTAVIDIN COATED BEADS

highly specific

DIFFERENCE TO GENOME CAPTURE

We want to visualize a genomic region

whereas here

we want to visualize the dna hybridised probe using a reporter

USES - BLOTTING LIKES PROBES

Northern blotting -DENATURE + ELECTROPHORESIS

DNA is denatured +separated by electrophoresis

DNA is hybridised with biotinylated probe

Fluorescent streptavidin beads used to visualise the probe

dna can be directly / indirectly labelled with fluorescent tags

High resolution hybridisation experiments

How can this be seen?

Indirect - reporter + affinity molecule

Direct = fluorescein / rhodamine - DNA LABELLED WITH NUCLEOTIDE

Attached to the nucleotides bound to the dna template

Fluorescence microscopy

-Excitation filter excites the fluorescent labelled probe

Imaging of fluorescent signals

-Equipment with APPROPRIATE EXCITATION SOURCE

-Excitation filter - wavelength of the fluorophore of interest is chosen

mages the hybridisation experiment

fluorescence microscopy

FISH

Fluorescence insitu hybridisation

-High resolution imaging microscopy

-dna probes with fluorescent marker - hybridised to sequence of interest

visualisation using fluorescence microscopy

• different colour probes used for different sequences

re-annealing of single stranded dna is also dependent on?

Concentration

fixed temperature of 65 degrees celciuus

salt concentration of 0.3M NACL

High concentration of DNA = faster kinetics of re-association more complementary strands available for pairing

Cot curve - concentration x time

Types of sequences

Unique sequences - appear ONCE IN THE GENOME

-Reassociate quickly - do not have as many strands to form repetitive regions

Repetitive sequences - Reassociate slowly

-Many copies of the same sequence are available for reassociation

They appear LATER IN THE COT CURVE

SLOWER reassociation rate

CoT curves

-What is measured

-What is the rate limiting step?

DENATURE + REANNEAL

-Shearing dna to 1.000 bp length

denaturing the dna with heat

lowering the temp to allow re annealing

Measure the % still single stranded at various time points

Rate limiting step?

-Collision of 2 complementary molecules - (formation of the double strand)

2nd order kinetics - rate of the association = initial concentration x time

reannealing - producs a Sigmodial curve - S SHAPED CURVE

-Characteristics of CoT analysis

S shaped curve

-Early on - conc of single stranded DNA is high

Later on - conc of single stranded dna is much lower

CoT ½ value - the point where ½ of the DNA is still single stranded

Unique sequences - CoT 1/2 value is much lower - faster reannealing

Repetitive sequences CoT ½ sequence is much higher - slower reannealing

CoT curve in the human genome

Complex

Not simple sigmoid

3 SECTIONS

Highly repeated DNA - about 50,000 copies - high CoT

Moderately repeated DNA 500 copies - lower CoT

Unique dna - 10 copies - Lowest CoT

-Each component has its own CoT ½ value - represents a characteristic portion of the genome

Highly repetitive elements of DNA are known as?

Use in Fish?

Highly repetitive DNA = CoT 1 DNA SUPPRESSOR

IN FISH

CoT 1 DNA

Prevents the probe from binding to repetitive DNA sequences

competes with the probe for binding sites

Improves signal-noise reatio

REDUCES THE BACKGROUND SIGNAL

IN FISH

Cot 1 DNA - added to solution before the probe

EASIER TO DETECT TARGET SEQUENCES

Microarray hybridisation

MICRO array

-grid of Immobilised probes in an array

High- throughput technique

-SOLUTION WITH SINGLE STRANDED DNA SEQUENCES

IMMOBILISED PROBES ASSAY IN GRID FORMAT

simultaneous analysis of thousands of sequences

Hybridising ARRAY of test sample nucleic acids to ARRAY OF MMOBILISED PROBES arranged in a grid

Each probe is specific to a sequence

Aqueous solution containing a Heterogenous collection of LABELLED DNA FRAGMENTS OR RNA TRANSCRIPTS

Denatures + allowed to hybridize with the probes on the array

Some probes - will have lots of sequences complementary to the dna sequences in the solution -

-other probes - few complementary sequences in the test sample - WEAK HYBRIDIZATION SAMPLE

Array comparative genomic hybridisation - ACGH

Reference DNA sample + test patient - labelled with different fluorophores

Fragmented using restriction endonucleases

-Denatured - made single stranded

Mixed in equal genomic amount + ALLOWED TO HYBRIDISE to the microarray containing KNOWN GENOMIC SEQUENCES (probe spot)

reference dna and the patient dna COMPETE FOR each probe spot

Reference = green, tested patients dna = red

Colour of the cell of the array AFTER hybridisation - measure of the relative amounts of the dna in each spot of the array

Yellow = even mix of the 2 - 2 normal copies of DNA in the sequence (green and red makes yellow)

Red = duplication - gain of genetic material e.g. 3 copies of a chromosome

green = deletion - loss of genetic material (the reference dna dominates) e.g. 1 copy of a chromosome

TESTING FOR GENETIC DISORDER, COPY NUMBER VARIATION, CANCER DIAGNOSTICS

PCR

Polymerase chain reaction

-Design of primerr forward and back - annealing of the primers

amplification of the primers

-make sure the primers dont sit in regions of highly repetitive DNA

-want to avoid repetitive elements in the genome

Taq polymerase - heat stable is used

denaturation, annealing, extension

Template dna, primers - short synthetic single strands

taq polymerase

buffer and mg

Primer design

what is important - how can we fix this?

-Want to avoid primers in regions of highly repetitive dna

dont want to amplify already highly repetitive dna

FOCUS ON THE UNIQUE DNA SEQUENCE

REPEAT MASKER

-Identifies repetitive elements + removes them or masks them

dna cloning can be done using?

Plasmid vectors

• cut the plasmid vector with a restriction enzyme

• replace the section in the plasmid with the human DNA to be cloned - using dna ligase to join them together

• transformation - bacteria grown in a media where ONLY THE RECOMBINANT PLASMIDS CAN SURVIVE

  antibiotic resistance in plasmids

• amplification of dna

BACTERIA CULTURED IN LARGE QUANTITIES

BAC

Making bac

BAC LIBRARIES

bacterial artificial chromosome vectors

Vector based on the

BAC VECTORS - enormous capacity

MBO 1 - cuts up the DNA of interest into fragments

Compatible “sticky ends” with Bam H I of the plasmid vector

genome projects - cloning of large regions of dna

Large inserts possible

DNA ISOLATED

DNA fragmented using restriction enzymes - MBo 1 -

then undergo pulsed gel electrophoresis

-dna fused to plasmid with dna ligase - mbo 1 sticky end to Bam HI

blue white screening - identifies colonies with INSERTS

Antibiotic selection - only bacteria with plasmid survive

-Robotic arrays speed up the process - can represent 90% of the genome

each 4×4 box - 8 clones plated in duplicate

summary

-comparative array genomic hybridisation

micro array hybridiation (immobilised probes)

cot1 dna - supressor of background noise for (repetitive dna)

SOUTHERN BLOT - FOR DNA

Restriction - electrophoreisis -hybridisation

What do genetic maps rely on?

Principle of genetic maps - genes located closely on the same chromosome are expected to be inherited together.

RFLP

1st genetic maps - constructed using polymorphic dna markers

How is polymorphism detected now?

-Restriction fragment length polymorphism - to detect polymorphic markers

A polymorphic marker is a region of DNA that varies between individuals (e.g., a mutation changing GATC → GACC). - detected by restriction enzymes (fragments)

Detect variation

MBOL - detects normal sequence GATC

-Allele 1 - has cut at GATC - Mbol cuts here

Allele 2 - has variation - not the normal GATC sequence - Mbol can’t cut here

In the past

-difference would be highlighted using a SOUTHERN BLOT ASSAY

-Mbol digests

fragments separated by electrophoresis

dna probe used to find the desired sequences

If has the restriction enzyme sequence - 2 fragment appears (cut at this site)

if not there is no cut

Now polymorphism detected using PCR

Cut using restriction eznymes - separate the fragments by electrophoresis

393 RFLP used for genetic linkage map of the human genome

AMPLIFICATION OF POLYMORPHISMS USING PCR

DIGESTION USING RESTRICTION ENZYME mbol

Second generation human genetic map - based on microsatellite DNA

Microsatellite DNA - aka Short tandem repeats

Instability arises - DURING MISTAKES IN REPLICATION

Polyacrylamide gel electrophoresis is used - see VARIATION IN SATELLITE DNA

-High resolution for small fragments of DNA

Separation based on size

-NUMBER OF REPEATS VARIES BETWEEN INDIVIDUALS - Good for genetic finger printing

Assayed by PCR - CREATE LARGE AMPLIFICATIONS TO TEST FOR polymorphisms in the satellite dna

Polymorphism = variation that occurs in the dna sequences among individuals in a population

Amplify using PCR

Separation for analysis using PAGE

Weissenbach et al - segregation analysis of 814 polymorphic locai containing (C-A)n repeats from 8 large families

Analysis of the sequences of dna

FORENSIC dna profiling

dna testing