3. DNA profiling

INTRONS

• DNA made of both introns and exons

• intron is a portion of a gene that does not code for AAs

• when mRNA is made during transcription the introns have to be removed before translation can happen- splicing

EXONS

• leaves only the exon parts which contain mRNA codons

• code for AAs so are used to make proteins

MINI AND MICRO SATELLITES

• repetitive DNA sequences that appear in a huge proportion of genomic DNA

  • also called variable number tandem repeats (VNTRs)

• distinguished from each other by no. of base pairs in the sequences

MINI SATELLITES

• has a DNA repeating sequence which is 10 to 60 base pairs in length

• can be repeated between 10 and 1000 times and this varies widely among individuals

• rich w/ G and C nucleotides

MICRO SATELLITES

• section of DNA which has simple sequence repeats of 1 to 9 base pairs long

• often referred to as short tandem repeats (STRs)

• rich w/ A and T nucleotides

DNA PROFILING

• also known as genetic or DNA fingerprinting

• compares introns (non-coding regions) and exons (coding regions) w/in DNA

• compares repeated sequences (mini- and micro- satellites) within introns

• same satellites appear on pairs of homologous chromosomes but no. of repeats could be diff as chromosomes may be maternal or paternal

• more closely related two individuals= more similar DNA

HOW DNA PROFILE IS MADE

1. DNA sample extracted- could be from body fluids, tissue samples or a mouth swab

2. DNA is amplified using PCR

3. fragments of diff lengths are produced by cutting up DNA using restriction endonuclease enzymes

4. fragments are separated and visualised using gel electrophoresis

DNA PROFILING- CRIME

• DNA Fingerprint is simply a ‘pattern’ produced on a piece of photographic film

• can we say 100% that suspect 1 committed the crime?

  • no fingerprint just establishes whether a suspect is likely to have been present at a crime scene

STEP 1- DIGESTION

• (PCR is completed first to make multiple copies of DNA)

• DNA sample is digested i.e. cut into fragments by restriction endonuclease enzymes

• cuts DNA at a specific recognition sites close to diff VNTRs (mini and micro satellite) sequences

  • DNA fragments produced will be diff lengths depending on how many repeats satellites contain

STEP 2- SEPARATION

• DNA fragments separated by gel electrophoresis

• loaded into a ‘well’ in agarose gel containing a buffering solution to maintain pH

• gel contains a dye which binds to DNA fragments

  • fluoresces when placed under UV light

STEP 3- SOUTHERN BLOT

• alkaline buffer is added to the gel and nylon filter paper is placed over it

• dry absorbent paper is used to draw the solution containing DNA from gel to nylon filter leaving them as ‘blot’

• pos charged Nylon binds DNA irreversibly under alkaline conditions

  • will be permanently in the same relative position they occupied on gel

• alkaline solution also denatures DNA fragments so strands separate and base sequences are exposed

• needs to happen as gel is too fragile handle

STEP 4- ADDITION OF PROBES

• as DNA is now single stranded, large quantities of gene probes are now added

• bind w/ complimentary DNA strands in a process called hybridisation

• gene probes are short pieces of DNA that have complimentary sequences to parts of DNA being sought

• each probe is labelled w/ a fluorescent molecule or radioactive isotope

• DNA profile can then be produced as a graph with each peak representing no. of repeats at each VNTR

IDENTIFYING INDIVIDUALS- FORENSICS

• to develop a DNA profile in criminal investigations, gene probes used to pick out short tandem repeats (micro-satellites widely used in DNA identification)

• the more micro-satellites used to make up a DNA profile the more accurate it will be

• family members are more likely than unrelated individuals to have micro-satellites in common

• statistically, chances of two people matching on 11 or more sites is so small that it is counted as reliable evidence in court

IDENTIFYING INDIVIDUALS- PATERNITY

• specific micro satellite markers are used to make matches between the child's DNA and potential fathers

• each child's micro satellite has to have come from either their father or mother

• if some come from neither then paternity is disproved