Molecular genetics

nucleoside

sugar + base

nucleotide

sugar + base + phosphate(s)

purines

adenine and guanine

pyrimidines

cytosine + uracil + thymine

molecule of nucleotide and bonds within

structure of DNA

• Chain of deoxyribonucleotides - ACTG

• Sugar phosphate backbone = sugars connect bases between 3' 5' carbons

• Two polynucleotide chains run anti-parallel and pair up AT  CG

• Chains twist into double helix

double helix and its qualities to account for key functions of DNA

Replication = strict base pairing gives simple mechanism for making exact copy

Storage of info = order of bases form triplet genetic code (codon) carrying instructions to make specific protein

Stability = strong phosphodiester bonds keep sequence intact (weak H bonds allow message to be read when necessary)

gene

Genes are ordered sequence of nucleotides (DNA) that encodes a specific functional product - RNA or PROTEIN

3 types of RNA

• rRNA and tRNA functional molecules in their own right

• mRNA - immediate molecule carrying the instructions for making a specific protein

DNA --> mRNA --> Protein

where is genetic variation

At molecular level, visible variation is due to differences in our proteins which in turn is due to differences in our DNA sequence

To study genetic variation we can analyse proteins or DNA

what is genetic variation

Alterations to DNA sequences can stop or alter protein function --> disease / altered characteristics

Studying protein reveals underlying genetic variation (polymorphism)

what changes don’t alter protein coded for

• Redundant changes

Both codons code for same amino acid e.g. CGC and AGG for arginine

• 3rd base wobble

e.g. GGG GGA GGT GGC code for glycine

types of mutation - change in DNA sequence

- deletions

-duplications

-inversions

-insertions

-translocations

-substitutions

Can involve any number of nucleotides from single bases to large sections of chromosome

errors in DNA replication can cause mutations as follows:

errors in recombination (crossing over) during meiosis can cause mutations as follows:

• Inversions, insertions or deletions between homologous chromosomes

• Translocations between non-homologous chromosomes

meiosis and homologous chromosomes during crossing over

mistakes when crossing over can lead to which types of mutations?

insertions and deletions and duplications

visual of crossing over

visual of crossing over leading to mutations

DNA analysis methods

If there are changes that do not alter a protein we need to analyse the genetic material (DNA) directly

1. DNA extraction

2. Identify the different variants (alleles)

3. Detect the results

where in a eukaryotic cell can DNA be extracted from?

three main steps of DNA extraction

1. Cell lysis

• A combination of heat, changes of pH, chemicals (e.g. detergents) and enzymes (proteases) are used to target proteins in cell membrane

2. Remove proteins

• Enzymes, chemicals and separation by centrifugation can do this

3. Recover the DNA

• Done by precipitation (DNA is insoluble in alcohol) or the use of specialised compounds that bind to the DNA

gel electrophoresis

• DNA extracted and placed and placed in gel

• Genomic DNA or isolated fragments can be analysed using electrophoresis

• Gel matrix acts like sieve to separate fragments

examples of gels used in gel electrophoresis

Agarose (AGE)

Polyacrylamide (PAGE)

SDS-PAGE

electrophoresis

AGE or PAGE can be used to view DNA

DNA has overall -ve charge

• Will move to anode

• Small fragments = faster

• Detect by staining with fluorescent dyes

distance travelled is …

equal to the length of DNA molecule (base pairs)

brightness of band is…

the amount of DNA present (ug)

locus

Specific point or location on chromosome (plural = loci)

allele

Different variations found at particular locus

polymorphism

Occurrence of more than one allele at a locus

genotype

Combination of alleles at locus

types of polymorphism - sequence variation

 base substitutions e.g. SNPs - single nucleotide polymorphisms

types of polymorphism - length variation

e.g. repetitive sequences

VNTRs - variable number tandem repeats

STRs - short tandem repeats

tools for molecular genetics - restriction enzymes

• Type ii restriction endonucleases

-enzymes that cut within a molecule of nucleic acid

-each enzyme has specific recognition sequence

• Polymorphisms analysed using restriction enzymes are known as RFLPs

Restriction Fragment Length Polymorphisms

each restriction enzyme has a specific recognition sequence for where they need to cut

length variation can be detected e.g. analysis of VNTRs

VNTRs

Enzyme cuts wither side of repeat region

Different DNA fragments are separated by electrophoresis

Length of fragments depends on number of repeats between two restriction sites

One band - homozygous

Two band - heterozygous

changes in DNA sequence (SNPs) can alter restriction enzyme recognition sites

SNPs

DNA is digested with enzyme

Different DNA fragments are separated by electrophoresis

Number of bands depends on how often restriction enzyme cuts - revealing underlying genetic variation

looking at SNPs using restriction enzymes

PCR - polymerase chain reaction - history

• Kary Mullis first described PCR in 1985, he won Nobel prize for Chemistry in 1993

• PCR allows us to isolate and copy a specific section of DNA

PCR reagents

• Template DNA - to be amplified

• dNTPs (deoxyribonucleotide triphosphates) - building blocks of DNA

• Buffers - maintains optimum pH

• DNA polymerase - the enzyme that synthesises DNA

• DNA primers - define the region to be amplified - short single stranded bit of DNA , marks start and end point of sequence wanted to copy

• Magnesium - cofactor required by enzyme

PCR process

forensic DNA analysis

• All cells (except RBCs) contain nucleus so could provide DNA for analysis

• PCR is very sensitive - only trace amounts of material needed

Forensic DNA analysis of STRs

Perfect for PCR based analysis

Similar structure to VNTR

But these are very short - 2-6 bases

DON’T use restriction enzymes - use PCR primers and PCR to copy

forensic DNA analysis of STRs

• Copy repeat region (using PCR)

• Length of each fragment depends on number of repeats - revealing the underlying genetic variation

• Separate the fragments using electrophoresis and measure the lengths

• Identify the alleles a person carries and their genotype

forensic DNA analysis of STRs

Don’t tend to use agarose gels but rather electronic machinery

Produce peaks

1 peak = 1 band on gel

forensic genetics - from crime scene to court

• Identification of particular genetic variants (alleles) present in a suspect and scene of crime evidence

• Is there a match?

• Apply population genetic theory to give courts a measure of probability

Missing person and DVI

• Identify matching body parts

• Identify individual by matching DNA to personal items

• Identify individual by comparing DNA to a known relative

paternity testing

• Mendelian genetics determines that a child must share one allele with each parent

• Genetic testing can include or exclude a tested man from being the biological father of a child

mendelian genetics

Molecular genetic analysis allows us to reveal variation in our DNA

Mendelian genetics determines patterns of inheritance  - a child must share one allele with each parent

Combo of alleles you inherit (genotype) determine characteristics expressed (phenotype)

population genetics

Q: how likely is it that the DNA match/paternity could be attributed to someone other than suspect?

When we present DNA evidence in court we need to understand probabilities

This requires estimates of allele/genotype frequency and the application of population genetic theory